Note: Descriptions are shown in the official language in which they were submitted.
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A METHOD FOR DESIGNING AND SCREENING RANDOM LIBRARIES OF
COMPOUNDS
FIELD OF THE INVENTION
The present invention relates generally to methods for generating and
screening Multidimensional Libraries (MDL) for proteins, polypeptides, and/or
peptides designated multidimensional peptides (MDPs) that are members of the
MDL,
for binding specificity and desired affinity for target molecule.
BACKGROUND OF THE INVENTION
In numerous fields, such as medicine and agriculture to name only a few, there
is an increasing need to find new molecules that can effectively modulate a
wide range
of biological processes. Traditional methods utilize "irrational drug design" -
the
process of selecting the right molecules from large ensembles or repertoires.
Generally, such methods include screening collections of natural materials,
such as
fermentation broths of plant extracts, or libraries of synthetic molecules,
with assays
that range dramatically in complexity from simple binding reactions to
elaborate
physiological preparations. Often, these assays provide only lead compounds,
which
require much improvement and refinement by empirical methods or by chemical
design
before any efficacious compound is identified. The process is time-consuming
and
costly, but it is unlikely to be totally replaced by rational methods even
when they are
based on detailed knowledge of the chemical structure of the target molecules.
Moreover, irrational drug design methods require continuous improvement in
both the
generation of repertoires and in the methods of their selection. Kay et al.,
Gezze
128:59-65 (1993).
Recently, several developments have been made in using peptides or
nucleotides to provide libraries of compounds for lead discovery. Generally,
there are
two different approaches to the construction of random peptide libraries. In
one
approach, peptides are chemically synthesized izz vitz-o in several formats.
For example,
the standard serial process of stepwise search of synthetic peptides now
encompasses a
variety of highly sophisticated methods in which large arrays of peptides are
synthesized in parallel and screened with acceptor molecules labeled with
fluorescent
or other reporter groups. The sequence of any effective peptide can be decoded
from
its address in the array. (Geysen et al.. Proc. Natl. Acad. Sci. USA 81:3998-
4002
CONFIRMATION COPY
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(1984); Maeji et al., J. Irnmunol. Methods 146:83-90 (1992); and Fodor et al.,
Science
251:767-775 (1991)).
In another approach, combinatorial libraries of peptides are synthesized on
resin beads. Each resin bead contains about 20 pmoles of the same peptide. The
beads
are then screened with labeled acceptor molecules. Those with bound acceptors
are
submitted to visual inspection and then physically removed. The peptide is
identified
by direct sequence analysis. Lam et al., Nature 354:82-84 (1991). Although
this
method may, in principle, be used with other chemical entities, it would
require
sensitive methods for sequence determination, thus making its use with other
chemical
entities very inefficient.
A different method of solving the problem of identification using a
combinatorial peptide library involves the use of hexapeptides. Houghten et
al., Nature
354:84-86 (1991). In particular, with hexapeptides of the 20 natural amino
acids, 400
separate libraries are synthesized, each of which has its first two amino acid
residues
fixed, i-e., invariant. The remaining four positions of the hexapeptides are
occupied by
all possible combinations. An assay based on competition for binding or other
activity
is then used to fmd the library with an active peptide. Once the library with
the active
peptide is located, 20 new libraries are synthesized and assayed in order to
determine
the effective amino acid residue in the third position. This process is then
repeated
until all six positions in the peptide are identified. However, this method is
inherently
time consuming and inefficient. Moreover, the size of the peptides that can be
assayed
is limited to six amino acid residues. Thus, its ability to assay the effect
of a protein's,
polypeptide's or peptide's secondary or tertiary structure on binding with a
target
molecule is extremely limited.
Recently, another approach using hexapeptides was suggested. In particular,
starting with 20 amino acids, a total of 120 (20 x 6) peptide mixtures are
synthesized.
In the 20 mixtures, position 6 contains a unique amino acid, and positions 1-5
contain a
mixture of all natural amino acids. In another 20 mixtures, position 5
contains a unique
amino acid and all other positions contain a mixture of all twenty amino
acids, etc.
Once synthesized, the 120 peptide mixtures are tested simultaneously and the
most
active of each of the 20 mixtures representing each position is identified.
Houghten,
Abstract, European Peptide Society 1992 symposium, Interlaken, Switzerland.
Although this method increases the speed in which an active peptide can be
found, it
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also possesses the inherent limitation of assaying hexapeptides. Thus, like
the method
explained above, the tertiary structure of compounds having activity can not
be
adequately explored.
A second approach using recombinant DNA techniques involves expressing
peptides i>z vivo as either soluble fusion proteins or viral capsid fusion
proteins. In
particular, a number of peptide libraries use the MI3 phage. MI3 is a
filamentous
bacteriophage that has been a used extensively in molecular biology
laboratories for the
past 20 years. The viral particle comprises six different capsid proteins and
one copy
of the viral genome, as a single-stranded circular DNA molecule. Once the M13
DNA
penetrates into a host cell such as E. coli, it is converted into double-
stranded, circular
DNA. The viral DNA carries a second origin of replication that is used to
generate the
single-stranded DNA found in the viral particles. During viral morphogenesis,
there is
an ordered assembly of the single-stranded DNA and the viral proteins, and the
viral
particles are excluded from cells in a process much like secretion. The M13
virus is
neither Iysogenic nor Iytic like other bacteriophages (i-ee. bacteriophage
~.). Once
infected, the cells chronically release the virus. This feature leads to
higher titers of
virus-infected cultures, i.e., 1012 pfulml.
The genome of the M13 phage is about 8000 nucleotides in length and has
been completely sequenced. The viral capsid protein, protein III (pIII) is
responsible
for the infection of bacteria. In E. coli, the F factor encodes the pillin
protein, which
interacts with the pIII protein, and is responsible for phage uptake. Hence,
all E. coli
hosts for the M13 virus are considered males because they carry the F factor.
Using
mutational analysis, investigators have determined that the 406 amino acid
long pIII
capsid protein has two domains. The C-terminus anchors the protein to the
viral coat,
while portions of the N-terminus of the pIII are essential for interactions
with the E.
coli pillin protein, Crissman and Smith, Virology 132: 445-455 (1984).
Although the
N-terminus anchors of the pIII protein have shown to be necessary for viral
infection,
the extreme N-terminus of the mature protein does not tolerate alterations. In
1985,
Smith published experiments reporting the use of the pIII protein of
bacteriophage M13
as an experimental system expressing a heterologous protein on the viral coat
surface,
Science 228:1315-1317 (1985). It was later recognized, independently by two
groups
that the M13 phage pIII gene display system could be a useful tool for mapping
antibody epitopes. Also de La Cruz et al. J. Biol. Chezzz. 263:4318-4322
(1988)] cloned
and expressed segments of the cDNA encoding the Plasmodium falciparuzzz
surface
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coat protein into the gene III, and recombinant phage were tested for
immunoreactivity
with a polyclonal antibody, Parmley and Smith Gene 73:305-318 (1988), cloned
and
expressed segments of the E. coli ~i-galactosidase gene in the gene III and
identified
recombinants carrying the epitope of an anti-~i-galactosidase monoclonal
antibody.
These authors also described a process termed "biopanning", in which mixtures
of
recombinant phage were incubated with biotinylated monoclonal antibodies, and
phage-antibody complexes specially recovered with streptavidin-coated plastic
plates.
In 1989, Parmley and Smith (Adv. Exp. Med. Biol. 251:215-218 (1989))
suggested that short, synthetic DNA segments cloned into the pIII gene might
represent
a library of epitopes. These authors reasoned that since linear epitopes were
often
about 6 amino acids in length, it should be possible to use a random
recombinant DNA
library to express all possible hexapeptides to isolate epitopes that could
bind to
antibodies.
Scott and Smith (Science 249:386-390 (1990)) described construction and
expression of an "epitope library" of hexapeptides on the surface of the M13
phage.
The library was made by inserting a 33 base pair Bgl I digested
oligonucleotide
sequence into the SfzI digested phage fd-tet, i.e., fUSE 5 RF. The 33 base
pair fragment
contained a random or "degenerate" coding sequence (NNK)6 where N represents
G, A,
T and C, and K represents G and T. The authors stated that the library
consisted of
2x108 recombinants expressing 4x10' different hexapeptides. Theoretically,
this
library expressed 69% of the 6.4x10' possible peptides (206). Cwirla et al.,
Proc. Nail.
Acad. Sci. U.S.A. 87:6378-6382 (1990) also described a somewhat similar
library of
hexapeptides expressed as gene pIII fusion of M13 fd phage. WO 91119818,
published
on Dec. 26, 1991 by Dower and Cwirla describes a similar library of pentameric
to
octomeric random amino acid sequences.
Furthermore, Devlin et al., Scieyace 249:404-401 (1990), described a peptide
library of about 15 residues generated using an (NNS) coding scheme for
oligonucleotide synthesis in which S is G or C.
Likewise, Christian et al., (J. Mol. Biol. 227:771-718 (1992)) have described
a
phage display library expressing decapeptides. The starting DNA was generated
by
means of an oligonucleotide comprising the degenerate codons (NN(G/T))lo with
a self-
complementary 3' terminus. In forming a hairpin, this sequence creates a self-
priming
replication site that could be used by T4 DNA polymerase to generate the
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complementary DNA strand. The double-stranded DNA was then cleaved at the SfiI
sites at the 5' terminus and hairpin for cloning into the fUSE 5 vector
described by
Scott and Smith Science 249:386-390 (1990).
These libraries may encompass a very large repertoire of different peptides
that
5 represent potential targets to a variety of macromolecules like receptors,
polypeptides,
enzymes, carbohydrates and antibodies. Therefore, phage display technology
appears
to be a very powerful tool for the selection of peptide sequences that bind to
a target
molecule. These peptides may find numerous applications, for example as
antigens in
vaccine composition, as enzyme inhibitors, as antagonists or agonists to
receptors.
However, due to the limited length of the peptides in the libraries discussed
above, the
peptides are not able to mimic native proteins, and adopt their conformation.
For
example, the monoclonal antibody 1B7, first described by Sato et al., Infect.
Irnnaun.
46:422-428 (1984) was initially raised against the B. pertussis toxin (PTX).
This
antibody is able to neutralize the toxin in vitro and to protect mice from
intracerebral
challenge with virulent B. pertussis. The epitope recognized by 1B7 was shown
to be
discontinuous and largely dependent on conformation. Hoping to obtain peptide
sequences that would mimic such a discontinuous epitope, Felici et al., Gene
128:21-27
(1993) constructed two phage display libraries consisting of nine random amino
acids
inserted in the major coat protein (pVIII), which nanopeptides are linear or
flanked by
two cysteine residues (circular). The two libraries were screened with the
antibody
1B7. The positive clones were sequenced and a consensus sequence was obtained
only
for linear peptides. In the absence of a three-dimensional structure of the
PTX
however, it was very difficult to determine how the consensus peptide sequence
corresponded to amino acid residues of the original protein that are important
in the
constitution of the discontinuous epitope recognized by the antibody 1B7.
Despite this
lack of information, the authors were expecting that the selected nanopeptides
would
sufficiently mimic the binding site of the original protein to serve as
antigens in the
production of vaccines against PTX. However, contrary to their expectations,
the
peptides were able to compete with PTX for the binding site of 1B7, but were
not
capable of sufficiently mimicking the discontinuous epitope of PTX to elicit
the
production of antibodies specific to the original antigen, PTX. Moreover, it
was
determined that the phage recombinant peptides adopted a conformation that may
be
governed by the surrounding phage sequences, which conformation is
recognizable by
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the antibody IB7. Thus, when peptides alone were synthesized without the
presence of
surrounding phage sequences, they lost their ability to bind the antibody 1B7.
The same group Luzzago et al., Gene 128:51-57 (1993) used the same libraries
to select oligopeptides that would bind to antibody H107, which recognizes the
native
conformation of the recombinant human ferritin H-subunit (H-Fer). This time
though,
the three-dimensional structure of H-Fer was known. The consensus peptide
sequence
obtained only for the linear selected peptides was used to assign to amino
acids
specifically located in the original protein, a putative role in the
conformation of the H-
Fer epitope. When the peptides were synthesized in the presence of surrounding
sequences located in the original protein as well as those synthesized with
surrounding
phage sequences, the peptides screened with H107 antibody were capable of
mimicking the original proteic assembly and efficiently bind the antibody
HI07.
These results indicate the unpredictability of libraries described above. In
particular, different results were obtained with peptides selected with two
different
antibodies. Thus, these libraries were not successful in the selection of
epitopes of all
existing antigens.
Yet another shorter peptide library is described by O'Neil et al., Proteins:
Structure, Fu>zction and Geyzetics 14:509-515 (1992). In this library, a
random circular
hexapeptide sequence was constructed and inserted it in the pIII phage
protein. The
library was then used to select targets to the receptor glycoprotein IIb/IIIa
(am,(33), a
member of the integrin family of cell adhesion molecules that mediate platelet
aggregation through the binding of fibrinogen and von Willebrand factor. The
purpose
of this work was to find targets that could be used as antagonists or as anti-
thrombotic
agents. The glycoprotein IIb/IIIa binds to a very short sequence commonly
known as
the RGD sequence. Using a circular library, the same authors were successful
in
identifying consensus sequence. The authors could indeed identify targets that
were
better antagonists than the target SK106760 (a cyclic peptide developed after
extensive
synthesis of an array of peptides) used to elute the phages of interest. They
also found
that a variant RGD sequence wherein the arginine was replaced by a lysine was
surprisingly one of the best anti-aggregatory selected peptides.
Kay et al. have also constructed the TSAR-9 library, which expresses 36
random amino acids at the N-terminus of the mature pIII molecule Geyze 128:59-
65
(1993). This library contains 10$ individual recombinants. While this value is
miniscule when compared to its potential coding diversity (2030, it is
biologically very
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diverse. For example, it has been estimated that only 105 40-amino acid exons
exist in
the human genome Dorit et al., Science 250:1377-1382 (1990). The library was
panned with streptavidin and a polyclonal goat anti-mouse IgG Fc antibody
preparation
coupled to paramagnetic beads. Streptavidin selected the class of phage
expressing the
amino acid motif, HPQ/M, similar to the motif identified by Devlin et al.,
Sciezzce
249:404-406 (1990) using a 15-amino acid random peptide library displayed on
phages. The polyclonal goat anti-mouse IgG Fc antibody preparation selected
phage-
displaying sequences similar to a region of the mouse IgG Fc. Thus, a single
immunodominant epitope on the mouse IgG was identified.
Other investigators have used other viral capsid proteins for the expression
of
non-viral DNA on the surface of phage particles. The protein pVIII is a major
viral
capsid protein and interacts with the single stranded DNA of M13 viral
particles at its
C-terminus. It is 50 amino acids long and exists in approximately 2,700
copies/particle. The N-terminus of the protein is exposed and will tolerate
insertions,
although large inserts have been reported to disrupt the assembly of fusion
pVIl1
proteins into viral particles, (Cesareni G., FEBS Lett. 307:66-70 (1992)). To
minimize
the negative effect of pVIII-fusion proteins, a phagemid system has been used.
Bacterial cells carrying the phagemid are infected with helper phage and
secrete viral
particles that have a mixture of both wild-type and fusion pVllI capsid
molecules.
Gene VIII has also served as a site for expressing peptides on the surface of
M13 viral
particles: 4 and 6 amino acid sequences corresponding to different segments of
the P.
falciparuzaz major surface antigen of the filamentous bacteriophage fd
(Greenwood et
1. J. Mol. Biol. 220:821-827 (1991)).
Leostra et al., J. Imnzuzzol. Methods 152:149-157 (1992), described the
construction of a library comprising annealing oligonucleotides of about 17 or
23
degenerate bases with an 8 nucleotide long palindromic sequence at their 3'-
ends to
express random hexa- or octapeptides as fusion proteins with the (3-
galactosidase
protein in a bacterial expression vector. The DNA was then converted into a
double-
stranded form with a Klenow DNA polymerase, blunt-end ligated into a vector,
and
then cloned into an expression vector of the C-terminus of a truncated [3-
galactosidase
to generate 107 recombinants. Colonies were then lysed, blotted on
nitrocellulose
filters and screened for immunoreactivity with several different monoclonal
antibodies.
A number of clones were isolated by repeated rounds of screening and then
sequenced.
However, as one of ordinary skill in the art can readily realize, it is
extremely
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laborious, and thus expensive to construct such a library. As a result, its
applications
are limited.
Pasquallni and Ruoslahti, Nature 380:364-366 (1996), reported an approach to
study organ-selective targeting based on ifa vivo screening of random peptide
libraries.
Peptides capable of mediating selective localization of phage to brain and
kidney blood
vessels were identified, and showed up to 13-fold selectivity for these
organs.
A particular "genetic" method of producing a library has been described
wherein the libraries are synthetic oligonucleotides themselves. Active
oligonucleotide
molecules are selected by binding to an acceptor site and then amplified by
the
polymerase chain reaction (PCR). PCR allows serial enrichment, and the
structure of
the active molecules is then decoded by DNA sequencing of clones generated
from the
PCR products. However, the repertoire is limited to nucleotides and the
natural
pyrimidine and purine bases, or those modifications that preserve specific
Watson-
Crick pairing and can be copied by polymerase, (Singer et al., Nucleic Acids
Res.
25:781-786 (1997)). Later this approach was further developed by the
introduction of
various chemical derivatives of nucleotides into already identified motives,
(Gold et al.,
Proc. Natl. Acad. Sci. U.S.A. 94:59-64 (1997)).
Another "genetic" method has been described in which the libraries of
synthetic oligonucleotides or DNA fragments encoding antibodies are displayed
in
ribosomes via an iu vitro translation system, (Hanes et al., Proc. Natl. Acad.
Sci. U.S.A.
94:4937-4942 (1997)); (He et al., Nucleic Acids Res. 25:5132-5134)). This
approach
allows effective expression of very large (up to 1015-101$ members) libraries,
while
phage display system is usually limited by 101°-1012 sequences. The
main advantage of
these genetic methods resides in the capacity for cloning and amplification of
the DNA
sequences, which allows enrichment by serial selection and provides a simple
and easy
method for decoding the structure of active molecules. Such results are not
available
when using libraries comprising peptides from a specific molecule, because, as
explained above, results obtained from such libraries are unpredictable since
the length
and the conformation of the exposed peptide may be sufficient to retrieve a
peptide
binding to a specific molecule, and yet not be suited to retrieve a peptide
that
efficiently mimics a more sophisticated binding region on another molecule.
However, a limitation of these genetic methods of producing libraries involves
the frequency of TAG (stop) codons in the oligonucleotides expressed by the
peptide
library. Efforts have been made to ameliorate this problem using hosts
carrying
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suppression. However, this strategy may not be 100% efficient to avoid stop
codon
expression in an oligonucleotide coding for a random peptide. Moreover, the
problem
becomes very serious when expressing oligonucleotides of longer length
encoding
random peptides.
Accordingly, what is needed are methods of generating libraries of peptides of
random and unspecified length, and comprising functional peptide units which
interact
with a target molecule, and structural peptide units, which help position the
functional
peptide units in order to maximize binding affinity for the target molecule.
What is also needed are methods of generating libraries of peptides of random
and unspecified length that utilize oligonucleotides of various and random
length.
What is also needed are methods of generating libraries of peptides wherein
the
peptides have random lengths, and are not limited to a certain length. As a
result, an
opportunity is made available to develop the secondary and/or tertiary
structures of the
potential binding peptides and in sequences flanking the actual binding
portions) of
the functional unit of the MDP. Such complex structural developments are not
feasible
when only oligonucleotides with fixed lengths are used.
What is further needed are libraries that can be advantageously screened to
identify multidimensional peptides (MDPs) having binding specificity for a
variety of
targets.
Moreover, what is also needed are methods of producing libraries comprising
oligonucleotides which are capable of expressing peptides of varying length,
and
effectively and efficiently minimize the negative impact of random stop codons
in
oligonucleotides of the library.
The citation of any reference herein should not be construed as an admission
that such reference is available as "Prior Art" to the instant application.
SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided a novel and useful
multidimensional library (MDL) for the selection of specific molecules that
bind or
interact in any way with molecules or molecular complexes (targets) of
interest. A
MDL may be represented by various natural or artificial polymeric compounds
including, but not limited, to isolated oligonucleotides, proteins,
polypeptides, peptides,
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polycarbohydrates, polyamines, heterocycles, or their combinations, to name
only a
few.
Broadly, the present invention extends to a multidimensional library for
screening molecules that potentially interact with a target molecule, wherein
the library
5 comprises at least one molecule comprising a general formula of (XYn)m,
wherein:
(XYn) is a repeating unit of the at least one molecule in which:
X is a functional unit that interacts with the target molecule,
Y is a structural unit,
10 n is the number of the structural units in the repeating unit, and
m is a number of repeating units in the at least one molecule.
In addition, the present invention extends to a multidimensional library as
described above, wherein the at least one molecule of the library is
detestably labeled.
Numerous detectable labels have applications herein, and can be readily
utilized by one
of ordinary skill in the art. Examples of detectable labels having
applications herein
include, but certainly are not limited to a radioactive element, a chemical
which
fluoresces, a chromophore, an enzyme, or an amplifiable nucleotide sequence,
to name
only a few. Particular examples of detectable labels having applications
herein are
described infra.
As explained above, the at least one molecule of a multidimensional library of
the invention can be comprised of numerous different types of molecules, e.g.,
oligonucleotides, proteins, polypeptides, peptides, polycarbohydrates,
polyamines,
heterocycles, or their combinations, to name only a few. In a particular
example
wherein the at least one molecule of the multidimensional library comprises a
protein, a
polypeptide, or a peptide, X is a functional peptide unit that potentially
participates in
an interaction between the at least one molecule and the target, Y is a
structural peptide
unit, n is an integer such that O~n-X10, and m is an integer, such that 220.
Moreover, as explained above, the at least one molecule of a multidimensional
library of the present invention can be an isolated oligonucleotide. In such
an
embodiment, the functional unit of the at least one molecule comprises a
nucleotide
regulatory sequence and the structural unit comprises a nucleotide sequence
comprising
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from 6 to at least 60 contiguous nucleotides. Numerous nucleotide regulatory
sequences having applications herein. Particular examples include, but
certainly are
not limited to a promoter, an enhancer, a cis-acting locus, a traps-acting
locus, an
attenuator, an upstream activator, or a regulatory non-translatable region
sequence.
Likewise, numerous promoters can serve as a functional unit in the at least
one isolated
oligonucleotide of a multidimensional library of the pxesent invention.
Particular
examples of such promoters include, but certainly are not limited to an SV40
early
promoter, a promoter contained in the 3' long terminal repeat of Rous sarcoma
virus, a
herpes thymidine kinase promoter, the regulatory sequences of the
metallothionein
gene, a [3-lactamase promoter, a tac promoter, an alcohol dehydrogenase
promoter, a
phosphoglycerol kinase promoter, an alkaline phosphatase promoter, an elastase
I gene
control region active in pancreatic acinar cells, an insulin gene control
region active in
pancreatic beta cells, an immunoglobulin gene control region active in
lymphoid cells,
a mouse mammary tumor virus control region active in testicular, breast,
lymphoid and
mast cells, an albumin gene control region active in liver, an alpha-
fetoprotein gene
control region active in liver, an alpha 1-antitrypsin gene control region
active in the
liver, a beta-globin gene control region active in myeloid cells, a myelin
basic protein
gene control region active in oligodendrocytic cells in the brain, a myosin
light chain-2
gene control region active in skeletal muscle, a gonadotropic releasing
hormone gene
control region active in the hypothalamus dihydrofolate reductas'e (DHFR)
promoter, a
constitutive RSV-LTR promoter, a metallothionein IIa gene promoter, a RSV-LTR
promoter, an immediate early promoter of hCMV, an early promoter of SV40, an
early
promoter of adenovirus, an early promoter of vaccinia, an early promoter of
polyoma, a
late promoter of SV40, a late promoter of adenovirus, a late promoter of
vaccinia, a late
promoter of polyoma, the lac system, the trp system, the TAC system, the TRC
system,
the major operator and promoter regions of phage lambda, a control region of
fd coat
protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or a
promoter
of yeast a mating factor.
Furthermore, the present invention extends to a multidimensional library
comprises at least one multidimensional peptide comprising a general formula
of
(XYn)m, wherein:
X is a functional peptide unit that participates in an interaction between the
at
least one multidimensional molecule and the target;
Y is a structural peptide unit;
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n is an integer, such that O~n~lO; and
m is an integer, such that 2-_<r~20,
wherein the at least one multidimensional peptide is encoded by at least one
isolated
oligonueleotide having a general formula of [(NNB)F~]m, wherein:
NisAorCorGorT/U;
B is C or G or T/U, but not A;
F is a codon encoding a predeternnined amino acid residue;
n is an integer, such that O~n~lO; and
m is an integer, such that 2~~20.
Naturally, the present invention extends to an isolated oligonucleotide which
encodes at least one molecule of a multidimensional library, wherein the
isolated
oligonucleotide comprises a general formula of [(NNB)F"]m, wherein:
NisAorCorGorT/U;
B is C or G or T/LJ, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~IO; and
m is an integer, such that 2~m~20.
Optionally, an isolated oligonucleotide of the present invention can further
comprise a signal sequence, which is described iyzfra, which signals the
unicellular host
to express the multidimensional peptide encoded by the isolated
oligonucleotide on the
surface of the unicellular host. Such signal sequences are well known to those
of
ordinary skill in the art, and can be spliced onto an isolated oligonucleotide
of the
present invention at the appropriate position using routine laboratory
techniques.
The present invention further encompasses numerous methods of synthesizing
such isolated oligonucleotides. One such method comprises synthesizing
oligonucleotides as described above, wherein N represents equimolar mixture of
A, C,
G, and T; B represents equimolar mixture of G, C, and T. Thus, the NNB motif
encodes any possible natural amino acids and contains only one stop codon
(TAG); F
represents a single pre-synthesized codon, a combination of several single
codons, or
their random pre-synthesized sequences that result in one or a combination of
pre-
selected annino acids; n is a number of codons resulting in structural blocks
of amino
acids which is a random value and could be for example, 0-10; m is a number of
functional codons which could be for example, 2-20.
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Another method encompassed by the present invention for synthesizing such
isolated oligonucleotides comprises using activated three-nucleotides
corresponding to
all natural amino acids as NNB codons and activated polynucleotides encoding
structural blocks of pre-selected amino acids as F codons.
Still another method encompassed by the present invention for synthesizing
isolated oligonucleotides having the general formula as described above
comprises
successive splitting and uniting steps, i.e., a "split-pull" synthesis. A
method for
performing a "split-pull" synthesis comprises the steps of:
(a) synthesizing three nucleotides having the NNB structure on a resin
support;
(b) dividing the resin support into n+1 fractions;
(c) continuing synthesis on each fraction of the resin support according to
the following scheme:
fraction 1: Resin-NNB
fraction 2: Resin-NNB-(N1N2N3)
fraction 3: Resin-NNB-(N1N2N3)2
fraction 4: Resin-NNB-(N1N2N3)3
fraction n+1: Resin-NNB (N1N2N3)n
where N1, N2, and N3 are nucleotides resulting in the F codon described above;
(d) mixing the resin support fractions together and continuing the
synthesis as set forth in step (a) to produce a compound having a general
structure of
Resin NNB-(N1N2N3)0-n-NNB;
(e) repeating steps (a) through (d) m times until a stochastic collection of
oligonucleotides having a general formula of [NNB-(N1N2N3)0-n]m is obtained;
and
(f) detaching the oligonucleotides from the resin support fractions.
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The use of the "split-pull" synthesis avoids the use of synthesized
oligonucleotides rich in GC nucleotides that are often found in libraries
using an NNS,
NNK, and NNB formula for variant codons. Such oligonucleotides are difficult
to
assemble and sequence properly.
Naturally, the present invention further extends to a cloning vector
comprising
an isolated oligonucleotide which encodes a multidimensional peptide of a
multidimensional library of the present invention, and an origin of
replication.
Numerous cloning vectors have applications herein, including E. coli, a
bacteriophage,
a plasmid, and a pUC plasmid derivative, to name only a few. A particular
example of
a bacteriophage that has applications as a cloning vector comprises a lambda
derivative. Moreover, a plasmid cloning vector further comprises a pBR322
derivative,
and a pUC plasmid derivative further comprises a pGEX vector, a pmal-c vector,
or a
pFLAG vector.
The present invention extends to an expression vector for expressing an
isolated oligonucleotide which encodes a multidimensional peptide of a
multidimensional library of the present invention. An expression vector of the
present
invention comprises an isolated oligonucleotide comprising a general formula
of
[(NNB)F~]"" wherein:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~IO; and
m is an integer, such that 2~m~e20,
operatively associated with a promoter.
Numerous expression vectors have applications in the present invention.
Particular examples are described infra. Moreover, numerous promoters can be
used in
an expression vector of the present invention. Examples of such promoters
include, but
certainly are not limited to an immediate early promoter of hCMV, an early
promoter
of SV40, an early promoter of adenovirus, an early promoter of vaccinia, an
early
promoter of polyoma, a late promoter of SV40, a late promoter of adenovirus, a
late
promoter of vaccinia, a late promoter of polyoma, the lac system, the trp
system, the
TAC system, the TRC system, the major operator and promoter regions of phage
lambda, a control region of fd coat protein, 3-phosphoglycerate kinase
promoter, acid
phosphatase promoter, and a promoter of yeast a mating factor.
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In addition, the present invention extends to a unicellular host transformed
or
transfected with an expression vector comprising an isolated oligonucleotide
which
encodes a multidimensional peptide of a multidimensional library of the
present
invention, wherein the isolated oligonucleotide comprises a general formula of
5 [(NNB)F~]m, in which:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~el0; and
10 m is an integer, such that 2~m~20,
operatively associated with a promoter.
A large variety of unicellular hosts have applications in the present
invention,
e.g. E. coli, Pseudomonas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M,
COSI, COS7, BSC1, BSC40, BMT10 and Sf9 cells, to name only a few.
15 In another embodiment, the present invention extends to a method for
generating a multidimensional library (MDL) comprising at least one
multidimensional
peptide having affinity for a target molecule, wherein the at least one
multidimensional
peptide has a general formula of (XYn)m, wherein:
X is a functional peptide unit that participates in an interaction between the
at
least one multidimensional peptide and the target;
Y is a structural peptide unit;
n is an integer, such that O~n~tl0;
m is an integer, such that 2-~m~20.
Such a method comprises the steps of:
(a) providing at least one oligonucleotide having the general formula of
[(NNB)F~]"" wherein:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~lO; and
m is an integer, such that 2~tm~20;
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(b) inserting the at least one oligonucleotide into an expression vector,
such that the at least one oligonucleotide is operatively associated with
a promoter;
(c) transforming or transfecting a unicellular host with the expression
S vector; and
(d) culturing the unicellular host under conditions that provide for
expression of the at least one oligonucleotide to produce the at least
one multidimensional peptide having affinity for the target molecule.
Furthermore, the present invention extends to a method fox identifying a
molecule of a
multidimensional library that interacts with a target molecule, comprising the
steps of:
(a) generating a multidimensional library (MDL) comprising at least one
molecule comprising a general formula of:
(XYn)m
wherein (XYn) is a repeating unit of the at least one molecule in which:
X is a functional unit of the at least one molecule,
Y is a structural unit of the at least one molecule,
n is the number of the structural units in the repeating unit, such that
O~n~lO, and
m is the number of repeating units in the at least one molecule, such
that 2~m~20;
(b) contacting the multidimensional library with the target molecule; and
(c) detecting binding of the target molecule with the at least one molecule.
Optionally, the at least one molecule of the multidimensional library is
detectably labeled. Particular examples of detectable labels having
applications herein
are described infra.
As explained above, the at least one molecule of a multidimensional library of
the invention can be comprised of numerous different types of molecules, ~.,
oligonucleotides, proteins, polypeptides, peptides, polycarbohydrates,
polyamines,
heterocycles, or their combinations, to name only a few. In a particular
example
wherein the at least one molecule of the multidimensional library comprises a
protein, a
polypeptide, or a peptide, X is a functional peptide unit that potentially
participates in
an interaction between the at least one molecule and the target, Y is a
structural peptide
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unit, n is an integer, such that O~n-X10, and m is an integer, such that
2~m~20. In a
method for screening for identifying a molecule of a multidimensional library
that
interacts with a target molecule, wherein the multidimensional library
comprises at
least one multidimensional peptide, a skilled artisan can readily perform the
step of
S generating the library using routine solid phase protein synthesis methods.
Alternatively, the step of generating such a library in a screening method of
the present
invention also comprises the steps of:
(a) providing at least one isolated oligonucleotide having the general
formula of:
[(NNB)Fn]m, wherein:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that 0-~n~tl0; and
m is an integer, such that 2-_em~20;
(b) inserting the at least one isolated oligonucleotide into an expression
vector, such that the at least one isolated oligonucleotide is operatively
associated with a promoter;
(c) transforming a unicellular host with the expression vector; and
(d) culturing the unicellular host under conditions that provide for
expression of the at Ieast one oligonucleotide to produce at Ieast one
multidimensional peptide having affinity for the target molecule.
Methods of producing such isolated oligonucleotides are encompassed by the
present invention, and described above and infra.
2S Numerous promoters have applications in such a method of the invention.
Particular examples include, but certainly are not limited to an immediate
early
promoter of hCMV, an early promoter of SV40, an early promoter of adenovirus,
an
early promoter of vaccinia, an early promoter of polyoma, a late promoter of
SV40, a
late promoter of adenovirus, a late promoter of vaccinia, a late promoter of
polyoma,
the lac system, the trp system, the TAC system, the TRC system, the major
operator and
promoter regions of phage lambda, a control region of fd coat protein, 3-
phosphoglycerate kinase promoter, acid phosphatase promoter, or a promoter of
yeast
a mating factor, to name only a few. Furthermore, numerous expression vectors
have
applications in a method fox generating a multidimensional library (MDL)
comprising
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at least one multidimensional peptide having affinity for a target molecule.
Particular
examples of such expression vectors are set forth infra.
Likewise, numerous unicellular hosts have applications in a method for
generating a multidimensional library (MDL) of the present invention, a g., E.
coli,
Pseudomonas, Bacillus, Strepomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7,
BSC1, BSC40, BMT10 and Sf9 cells, to name only a few.
In another embodiment, the present invention extends to a kit for screening
molecules that potentially interact with a target molecule. Such a kit
comprises:
(a) a predetermined amount of a multidimensional library (MDL)
comprising at least one molecule that potentially has affinity for the
target molecule, wherein the at least one molecule has a general
formula of (XYn)m, wherein:
X is a functional unit that interacts with the target molecule;
Y is a structural unit;
n is an integer, such that O~n~lO;
m is an integer, such that 2-_en~20,
(b) other reagents; and
(c) directions for use of the kit.
Optionally, the at least one molecule of a multidimensional library of a kit
of
the invention is detectably labeled. Particular examples of such labels are
described
infra.
Furthermore, the present invention extends to a kit for screening molecules as
described above, wherein the at least one molecule comprises an isolated
oligonucleotide, a protein, a polypeptide, a peptide, a carbohydrate, a
polyamine, a
heterocyclic molecule, or a combination thereof. In a particular embodiment,
wherein
the at least one molecule comprises a protein, a polypeptide, or a peptide, X
is a
functional peptide unit that participates in an interaction between the at
least one
multidimensional molecule and the target, Y is a structural peptide unit, n is
an integer
such that O~n~lO, and m is an integer such that 2~n~20. Reagents having
applications
in this embodiment of a kit of the present invention are generally those that
maintain a
peptide's native conformation. Examples of such reagents include, but
certainly are
not limited to protease inhibitors, such as PMSF, phosphate buffered saline,
TRIS
glycine buffer, TRIS HCl buffer, etc., wherein the reagents are at
physiological pH.
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Other such reagents well known to those of ordinary skill in the art are
encompassed
herein.
In another embodiment, the present invention extends to a kit for screening
molecules of a multidimensional library that potentially interact with a
target molecule,
wherein the kit comprises:
(a) a unicellular host transformed or transfected with an expression vector
comprising at least one oligonucleotide operatively associated with a
promoter, wherein the at least one oligonucleotide has the general
formula of [(NNB)Fn]m, wherein:
N is A or C or G or T/LT;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~lO; and
m is an integer, such that 2-~rr~20;
(b) reagents for expressing the at least one oligonucleotide;
(c) other reagents; and
(d) directions for use the kit.
With such a kit, one of ordinary skill in the art can readily express the at
least
one isolated oligonucleotide inserted into the unicellular host to produce the
at least one
multidimensional peptide of a multidimensional library when needed. Then, this
library can readily be used to screen molecules of the library for interaction
with a
target molecule. Optionally, a signal sequence could be placed on the at least
one
multidimensional peptide using routine molecular biology techniques well known
to
those skilled in the art.
Reagents having applications herein include those that aid a protein maintain
its native conformation, such as those described above, as well as those used
to express
the at least one oligonucleotide inserted into the unicellular host.
Particular examples
of such reagents include, but certainly are not limited to PCR reagents, such
as
oligonucleotides, oligonucleotide primers, enzymes, gel matrixes, buffers,
etc.
Furthermore, the present invention extends a kit for screening molecules that
potentially interact with a target molecule, comprising:
(a) a predetermined amount of a multidimensional library (MDL)
comprising at least one isolated oligonucleotide that potentially has
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affinity for the target molecule, wherein the at least one molecule has a
general formula of (XYn)m, wherein:
X is a functional unit comprising a nucleotide regulatory sequence that
potentially interacts with the target molecule;
5 Y is a structural unit comprising a nucleotide sequence comprising
from 5 to at least 50 contiguous nucleotides;
n is an integer, such that O~n~lO;
m is an integer, such that 2~m~20,
(b) other reagents; and
10 (c) directions for use of the kit.
Numerous nucleotide regulatory sequences can serve as a functional unit in the
at least one molecule in a multidimensional library used to screen molecules
that
potentially interact with a target molecule. Particular nucleotide regulatory
sequences
15 having applications herein comprise a promoter, an enhancer, a cis-acting
locus, a
trans-acting locus, an attenuator, an upstream activator, or a regulatory non-
translatable
region sequence.
Moreover, the present invention extends to a multidimensional library (MDL)
comprising at least one multidimensional peptide having affinity for a target
molecule,
20 wherein the at least one multidimensional peptide has a general formula of
(XYn)m,
wherein:
X is a functional peptide unit that participates in an interaction between the
at
least one multidimensional molecule and the target;
Y is a structural peptide unit;
n is an integer, such that O~n-_<10; and
m is an integer, such that 2~m~20,
wherein such a libxary is made with a process comprising the steps of:
(a) providing at least one isolated oligonucleotide having the general
formula of:
[(NNB)Fn]"" wherein:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that 0-~n~10; and
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m is an integer, such that 2~m~20;
(b) inserting the at least one isolated oligonucleotide into an expression
vector, such that the at least one isolated oligonucleotide is operatively
associated with a promoter;
(c) transforming a unicellular host with the expression vector; and
(d) culturing the unicellular host under conditions that provide for
expression of the at least one oligonucleotide to produce at least one
multidimensional peptide having affinity for the target molecule.
Accordingly, it is an object of the present invention to provide a
multidimensional
library wherein the overall size of the construct as well as the number of the
functional
units and the structural units is limited only by the vehicle that is used to
display the
library.
It is another object of the present invention to provide a multidimensional
library (MDL) that enables a skilled artisan to select molecules with affinity
and
selectivity of interaction with a pre-selected target molecule.
It is another object of the present invention to provide a method for
identifying
a molecule interacting with a target of interest that is reproducible, quick,
simple,
efficient and relatively inexpensive.
It is another object of the present invention to provide methods of producing
multidimensional libraries using isolated oligonucleotides of random size with
a
minimal amount of internal stop codons. The limitation of stop codons becomes
especially important when the size of the inserted oligonucleotide is large,
e.g., greater
than about 20 codons. For example, using a heretofore known "genetic" method
for
producing peptide libraries, in an isolated oligonucleotide of 100 codons, the
possibility
of not having a stop codon, i-e., of having an open reading frame, would be
(47/48)100
or about 12%. However, using a method of the present invention, the
possibility of
having a stop codon in the reading frame would be (31/32)100 or about only 4%.
It is another object of the present invention to provide a method for
generating
and screening a large library of diverse proteins, polypeptides and/or peptide
molecules.
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It is yet another object of the present invention to provide a rapid and easy
way
of producing a large multidimensional library comprising a plurality of longer
proteins,
polypeptides and/or peptides that can be efficiently screened to identify
those having
novel and/or improved specificities, affinities and stabilities for a
particular target of
choice.
It is yet still another object of the present invention to provide a method
for
producing a multidimensional library as described above which avoids the need
for
purifying, or isolating genes, nor any need fox detailed knowledge of the
function of
portions of the binding sequence, the amino acids that are involved in target
binding
sequence, or the amino acids that are involved in target binding in order to
produce
MDP. Moreover, since MDPs are screened iyz vitro, the solvent requirements
involved
in MDP/target interactions are not limited to aqueous solvents; thus, non-
physiological
interactions and binding conditions different from those found in vivo can be
exploited.
It is still yet another object of the present invention to provide a method
for
designing variant oligonucleotides that permits greater variability in the
sequence of the
oligonucleotides than is presently permitted using schemes described above.
Moreover, non-natural amino acids could also be used in an MDL described
herein if an appropriate expression system is used supplied with tRNA modified
to
express respective non-natural amino acids [Satoh et al., Nucleic Acids S~nnp.
Ser.
37:117-118 (1997)].
These and other aspects of the present invention will be better appreciated by
reference to the following drawings and Detailed Description.
BRIEF DESCRIPTfON OF THE DRAWINGS
FIG. 1 is a schematical view of the construction of a linear oligonucleotide
library. (A) The vector fUSE 5 contains two non-complementary SfiI sites
separated by
a 14 base pairs "stuffer fragment". Removal of the SfiI fragment allows
oriented
ligation of oligonucleotides with the appropriate cohesive ends. (B) The
oligonucleotide ON-69 was annealed to two half site fragments to form cohesive
termini complementary to SfiI sites 1 and 2 in the vector. The gapped
structure, where
the single-stranded region comprises the variable 16-mer codon sequence was
ligated
to the vector and electro-transformed into E. coli.
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FIG. 2 is a schematical view of the strategy for the "split-pull" synthesis.
N1-
equimolar mixture of the 4 nucleotides ; NZ - mixture of G (19%), A (31%), T
(31%),
and C (19%) ; N3 - mixture of G (39%), T (22%) and C (39%).
FIG. 3 shows the amino acid sequences (deduced from the DNA sequences) of
the N-terminal peptides of pllI of infectious phages randomly selected from
the library.
Individual isolates were sequenced with the oligo primer fUSE32P, which is 32
nucleotides downstream of the gene III cloning site of fUSE 5. Structural
blocks are
underlined. Single letter code for amino acids is A (Ala), C (Cys), D (Asp), E
(Glu), F
(Phe), G (Gly), H (His), I (Ile), K (Lys), L (Leu), M (Met), N (Asn), P (Pro),
Q (Gln),
R (Arg), S (Ser), T (Thr), V (Val), W (Trp), Y (Tyr).
FIG. 4 depicts the amino acid frequencies in functional domains (A) and in
structural blocks (B) analyzed in randomly chosen isolates; 2X represent 100%
deviation from the optimal frequency that is equal to 5 % for functional
domain (A) and
is equal to 20% for structural block (B). Figure 4C shows block length
distribution.
FIG. 5 shows selection of phages that bind to streptavidin. Phages from MDL
were bound to strepavidin coated microtiter wells and then eluted with
glycinelHC1
buffer pH 2.2. Enrichment was calculated as the total number of phages
recovered after
the elution (n;, measured in transducing units) divided by the number of
transducing
units recovered after the first round of selection (n1). The data represents
mean values
from plating in triplicate (SEM < 10%).
FIG. 6 shows the amino acid sequences (deduced from DNA sequence) of the
N-terminal peptides of pIII of 29 clones recovered after 2, 3, or 4 rounds of
panning on
streptavidin coated microtiter wells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based upon the discovery that surprisingly and
unexpectedly, libraries comprising molecules of random length that are not
limited to a
maximum length can be constructed easily and efficiently. Moreover, such
libraries
can be advantageously screened to identify molecules of a multidimensional
library of
the present invention that possess binding specificity for a variety of
targets. Thus,
libraries of the present invention are novel, useful and unobvious with
respect to
heretofore known libraries in which the length of molecules of the library is
limited,
such as, for example, less than 15 and preferably about 10-12 amino acids.
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Broadly, the present invention extends to a multidimensional library for
screening molecules that potentially interact with a target molecule, wherein
said
library comprises at least one molecule comprising a general formula of
(XYn)m,
wherein:
(XYn) is a repeating unit of the at least one molecule in which:
X is a functional unit that interacts with the target molecule,
Y is a structural unit,
n is the number of structural units in the repeating unit, and
m is a number of repeating units in the at least one molecule.
The presence of structural and functional units in a molecule of a
multidimensional library of the present invention provide the opportunity for
the
development of secondary and/or tertiary structure in molecules, which
potentially
increase their affinity for the target, and more accurately mimic molecules
and
compounds found in vivo. Such complex structural developments are not feasible
in
libraries utilizing peptides of a limited length.
As explained above, An MDL of the present invention may be represented by
various natural or artificial polymeric compounds including, but not limited,
to isolated
oligonucleotides, e.g. oligonucleotides, proteins, polypeptides, peptides,
polycarbohydrates, polyamines, heterocycles, or their combinations, to name
only a
few.
For example, the present invention extends to a multidimensional library for
screening molecules that potentially interact with a target molecule, wherein
the library
comprises at least one molecule comprising a general formula of (XY")m, and
the at
least one molecule of the library comprises a protein, a polypeptide or a
peptide having
in which(XYn) is a repeating unit of the at least one molecule wherein:
X is a functional peptide unit that participates in an interaction between the
at
least one molecule and the target;
Y is a structural peptide unit;
n is an integer such that O~n~lO; and
m is an integer, such that 2-~m~20.
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In another example, the present invention extends to a multidimensional
library
for screening molecules that potentially interact with a target molecule,
wherein said
library comprises at least one isolated oligonucleotide comprising a general
formula of
(XYn)m, wherein:
5 (XYn) is a repeating unit of the at least one oligonucleotide;
X is a functional unit comprising a nucleotide regulatory sequence;
Y is a structural unit comprising a nucleotide sequence comprising from 6 to
at
least 60 contiguous nucleotides.
Moreover, the present invention extends to a multidimensional library
10 comprising at least one multidimensional peptide having affinity for a
target molecule,
wherein the at least one multidimensional peptide has a general formula of
(XYn)m,
wherein:
X is a functional peptide unit that participates in an interaction between the
at
least one multidimensional peptide and the target;
15 Y is a structural peptide unit;
n is an integer, such that O~n~lO;
m is an integer, such that 2~err~20,
wherein such a library is made by a process comprising the steps of:
(a) providing at least one oligonucleotide having the general formula of
20 [(NNB)F~]m, wherein:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a colon encoding a predetermined amino acid residue;
n is an integer, such that O~n~lO; and
25 m is an integer, such that 220;
(b) inserting the at least one oligonucleotide into an expression vector,
such that the at least one oligonucleotide is operatively associated with
a promoter;
(c) transforming a unicellular host with the expression vector; and
(d) culturing the unicellular host under conditions that provide for
expression of the at least one oligonucleotide to produce the at least
one multidimensional peptide having affinity for the target molecule.
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Furthermore, as explained above, the present invention extends to cloning
vectors and expression vectors comprising, iyzter alia, at least one
oligonucleotide
having the general formula of [(NNB)F~]m, wherein:
NisAorCorGorT/LT;
B is C or G or TIU, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~lO; and
m is an integer, such that 2~m~20
In addition, the present invention extends to unicellular hosts transformed or
transfected with vectors of the present invention.
Numerous terms and phrases used throughout the instant Specification and
appended Claims are defined below:
As used herein, the phrase "multidimensional library" or "MDL" refers to a
library of various natural or artificial polymeric compounds, including, but
not limited
to, polynucleotides, polypeptides, peptides, polycarbohydrates, polyamines,
heterocycles, or a combination thereof.
As used herein, the phrase "multidimensional peptide" or "MDP" refers to a
polypeptide or peptide having a general formula of (XYn)m, wherein X is a
functional
peptide unit that participates in the interaction between the multidimensional
peptide
and a target molecule, and Y is a structural peptide units) involved in
positioning the
functional peptide units) so as to maximize its/their interaction with the
target
molecule. The presence of structural and functional peptide units provide the
opportunity for the development of secondary and/or tertiary structure in the
potential
binding protein/peptides, and in sequences flanking the actual binding
portions) of the
binding domain of MDP. Such complex structural developments are not feasible
in
libraries utilizing peptides of a limited length.
MDPs or MDP composition comprising a part thereof may be used in any in
vivo or ifz vitro application that might make use of a peptide or a
polypeptide that
specifically interacts with a target. Thus, MDP or the MDP composition can be
used in
place of, or to bind to, a cell surface receptor, a viral receptor, an enzyme,
a lectin, an
integrin, an adhesin, a Cap binding protein, a metal binding protein, DNA or
RNA
binding proteins, immunoglobulins, vitamin cofactors, peptides that recognize
any bio-
organic or inorganic compound, etc.
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Selected MDPs that possess catalytic activities may be used as artificial
enzymes to chemically modify targets.
By virtue of the affinity for a target, MDPs or compositions comprising MDPs
or a portion thereof used ih vitro and in vivo can deliver a chemically or
biologically
active moiety, such as a metal ion, a radioisotope, peptide, toxin or fragment
thereof, or
an enzyme or fragment thereof, or pharmaceutical formulation thereof, to the
specific
target in or on the cell. The MDPs can also have an in vitro utility similar
to
monoclonal antibodies or other specific binding molecules for the detection,
quantification, separation or purification of other molecules. In one
embodiment, a
number of MDPs or the binding domains thereof can be assembled as multimetric
units
to provide multiple binding domains that have the same specificity and can be
fused to
another molecule that has a biological or chemical activity.
The MDPs that are produced with a method of the present invention can
replace the function of macromolecules such as monoclonal or polyclonal
antibodies
and thereby circumvent the need for the complex methods of hybridoma formation
or
an in vivo antibody production. Moreover, MDPs differ from other natural
binding
molecules in that MDPs have an easily characterized and designed activity that
can
allow their direct and rapid detection in a screening process. Furthermore, it
is
expected that some MDP molecules may possess catalytic activities and can
therefore
be used as artificial enzymes.
As used in the present invention, MDPs are intended to encompass a
concatenated protein, polypeptide and/or peptide that includes structural and
functional
elements. The affinity of the functional element of the MDP molecule for a
target is
characterized by: 1) its strength of binding under specified conditions; 2)
the stability
of its binding or other interactions under specified conditions; and, 3) its
selective
specificity for the chosen target. The structural element of the MDP molecule
is a
domain that separates the functional elements and locates them at the most
appropriate
coordinates relative to each other.
As used herein, the terms "target" or "target molecule" can be used
interchangeably, and refer to a substance, including a molecule or portion
thereof, or
complex of several molecules for which a receptor naturally exists or can be
prepared
according to the method of the invention. In particular, a target is a
substance that
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specifically interacts with the functional units) of a molecule of an MDL of
the present
invention, and includes, but is not limited to, a chemical group, an ion, a
metal, a
protein, a glycoprotein or any portion thereof, a peptide or any portion of a
peptide, a
nucleic acid or any portion of a nucleic acid, a sugar, a carbohydrate or a
carbohydrate
polymer, a lipid, a fatty acid, a vital particle or portion thereof, a
membrane vesicle or
portion thereof, a cell wall component, a synthetic organic compound, a bio-
organic
compound and an inorganic compound.
An MDP that can bind to a target can function as a receptor, i-e., a lock into
which the target fits and binds; or an MDP can function as a key which fits
into and
binds to a target when the target is a larger protein molecule; or an MDP can
function
as a catalyst to accelerate or to slow down the chemical or biochemical
conversion of
the target. In this invention, a target is a substance that specifically
interacts with, or
binds to, an MDP and includes, but is not limited to, an organic chemical
group, an ion,
a metal or non-metal inorganic ion, a glycoprotein, a protein, a polypeptide,
a peptide, a
nucleic acid, a carbohydrate or a carbohydrate polymer, a lipid, a fatty acid,
a viral
particle, a membrane vesicle, a Bell wall component, a synthetic organic
compound, a
molecular complex or any portion of any of the above.
As used herein, the singular forms "a," "an" and "the" include plural
referents
unless the context clearly dictates otherwise.
Moreover, in accordance with the present invention there may be employed
conventional molecular biology, microbiology, and recombinant DNA techniques
within the skill of the art. Such techniques are explained fully in the
literature. See,
e.~., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,
Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New
York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach,
Volumes
I and II (D.N. Glover ed. 1985); Oligonucleotide Syrzthesis (M.J. Gait ed.
1984);
Nucleic Acid Hybridization, B.D. Hames & S.J. Higgins eds. (1985);
Transcription And
Trarzslation, B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture ,
R.I.
Freshney, ed. (1986); Irnnzobilized Cells Arzd Erzzymes IRL, Press, (1986); B.
Perbal, A
Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.),
Current
Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). Therefore, if
appearing herein, the following terms shall have the definitions set out
below.
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A "vector" is a replicon, such as plasmid, phage or cosmid, to which another
DNA segment may be attached so as to bring about the replication of the
attached
segment. A "replicon" is any genetic element (e.~., plasmid, chromosome,
virus) that
functions as an autonomous unit of DNA replication ifz vivo, i.e., capable of
replication
under its own control.
A "cassette" refers to a segment of DNA that can be inserted into a vector at
specific restriction sites. The segment of DNA encodes a polypeptide of
interest, and
the cassette and restriction sites are designed to ensure insertion of the
cassette in the
proper reading frame for transcription and translation.
A cell has been "transfected" by exogenous or heterologous DNA when such
DNA has been introduced inside the cell. A cell has been "transformed" by
exogenous
or heterologous DNA when the transfected DNA effects a phenotypic change.
Preferably, the transforming DNA should be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell.
"Heterologous" DNA refers to DNA not naturally located in the cell, or in a
chromosomal site of the cell. Thus, oligonucleotides that encode
multidimensional
peptides of a multidimensional library, as well as oligonucleotides which make
up a
multidimensional library of the invention, are heterologous DNA when inserted
into a
vector that is used to transform/transfect a cell.
A "nucleic acid molecule" refers to the phosphate ester polymeric form of
ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules")
or
deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or
deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as
phosphorothioates and thioesters, in either single stranded form, or a double-
stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
The term oligonucleotide, and in particular DNA or RNA, or isolated nucleic
acid
molecule, refers only to the primary and secondary structure of the molecule,
and does
not limit it to any particular tertiary forms. Thus, this term includes double-
stranded
DNA found, inter alia, in linear or circular DNA molecules (~., restriction
fragments),
plasmids, and chromosomes. Tn discussing the structure of particular double-
stranded
DNA molecules, sequences may be described herein according to the normal
convention of giving only the sequence in the 5' to 3' direction along the non-
transcribed strand of DNA (i.e., the strand having a sequence homologous to
the
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mRNA). A "recombinant oligonucleotide" is a oligonucleotide that has undergone
a
molecular biological manipulation. '
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule,
such as a cDNA, genomic DNA, or RNA, when a single stranded form of the
nucleic
5 acid molecule can anneal to the other nucleic acid molecule under the
appropriate
conditions of temperature and solution ionic strength (see Sambrook et al.,
supra). The
conditions of temperature and ionic strength determine the "stringency" of the
hybridization. For preliminary screening for homologous nucleic acids, low
stringency
hybridization conditions, corresponding to a Tm of 55° C can be used,
e.g., 5x SSC,
10 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5%
SDS).
Moderate stringency hybridization conditions correspond to a higher Tm, e.g.,
40%
formamide, with 5x or 6x SSC. High stringency hybridization conditions
correspond
to the highest Tm, e.g., 50% formamide, 5x or 6x SSC. Hybridization requires
that the
two nucleic acids contain complementary sequences, although depending on the
15 stringency of the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the length of
the
nucleic acids and the degree of complementation, variables well known in the
art. The
greater the degree of similarity or homology between two nucleotide sequences,
the
greater the value of Tm for hybrids of nucleic acids having those sequences.
The
20 relative stability (corresponding to higher T"~ of nucleic acid
hybridization decreases in
the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than
100 nucleotides in length, equations for calculating Tm have been derived (see
Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic
acids, i-e.,
oligonucleotides, the position of mismatches becomes more important, and the
length
25 of the oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8).
Preferably a minimum length for a hybridizable nucleic acid is at least about
20
nucleotides; preferably at least about 30 nucleotides; more preferably the
length is at
least about 40 nucleotides; and even more preferably at least about 50
nucleotides.
In a specific embodiment, the term "standard hybridization conditions" refers
30 to a Tm of 55° C, and utilizes conditions as set forth above. In a
preferred embodiment,
the Tm is 60° C; in a more preferred embodiment, the Tm is 65°
C.
"Homologous recombination" refers to the insertion of a foreign DNA
sequence of a vector in a chromosome. Preferably, the vector targets a
specific
chromosomal site for homologous recombination. For specific homologous
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31
recombination, the vector will contain sufficiently long regions of homology
to
sequences of the chromosome to allow complementary binding and incorporation
of
the vector into the chromosome. Longer regions of homology, and greater
degrees of
sequence similarity, may increase the efficiency of homologous recombination.
A DNA "coding sequence" is a DNA sequence which is transcribed and
translated into a polypeptide in a cell ifz vitro or irz vivo when placed
under the control
of appropriate regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a translation stop
codon at
the 3' (carboxyl) terminus. A coding sequence can include, but is not limited
to
prokaryotic sequences, cDNA from eukaxyotic mRNA, genomic DNA sequences from
eukaryotic (~., mammalian) DNA, and even synthetic DNA sequences. If the
coding
sequence is intended for expression in a eukaryotic cell, a polyadenylation
signal and
transcription termination sequence will usually be located 3' to the coding
sequence.
Transcriptional and translational control sequences are nucleotide regulatory
sequences, such as promoters, enhaneers, terminators, and the like, that
provide for the
expression of a coding sequence in a host cell. In eukaryotic cells,
polyadenylation
signals are control sequences.
A "promoter" is a nucleotide regulatory region capable of I binding RNA
polymerase in a cell and initiating transcription of a downstream (3'
direction) coding
sequence. For purposes of defining the present invention, the promoter
sequence is
bounded at its 3' terminus by the transcription initiation site and extends
upstream (5'
direction) to include the minimum number of bases or elements necessary to
initiate
transcription at levels detectable above background. Within the promoter
sequence
will be found a transcription initiation site (conveniently defined for
example, by
mapping with nuclease S 1), as well as protein binding domains (consensus
sequences)
responsible for the binding of RNA polymerase.
A coding sequence is "under the control" of transcriptional and translational
control sequences in a cell when RNA polymerase transcribes the coding
sequence into
mRNA, which is then traps-RNA spliced and translated into the protein encoded
by the
coding sequence.
A "signal sequence" can be included at the beginning of the coding sequence of
a protein, polypeptide or peptide that is to be expressed on the surface of a
cell. This
sequence encodes a signal peptide, N-terminal to the mature polypeptide, that
directs
the host cell to translocate the polypeptide. The term "translocation signal
sequence" is
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used herein to refer to this sort of signal sequence. Translocation signal
sequences can
be found associated with a variety of proteins native to eukaryotes and
prokaryotes, and
are often functional in both types of organisms.
As used herein, the phrase "nucleotide regulatory sequence" refers to
nucleotide sequences that are involved in the regulation of expression of a
particular
gene.
As used herein, the term "promoter" refers to a nucleotide regulatory sequence
capable of binding RNA polymerise in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining the
present
invention, the promoter sequence is bounded at its 3' terminus by the
transcription
initiation site and extends upstream (5' direction) to include the minimum
number of
bases or elements necessary to initiate transcription at levels detectable
above
background. Within the promoter sequence will be found a transcription
initiation site.
As used herein, the term "enhancer" refers to a nucleotide regulatory sequence
that increases the transcriptional activity of nearby structural genes.
Enhances can act
over a distance of thousands of base pairs and can be located 5' or 3' to the
gene they
affect.
As used herein, the phrase "cis-acting locus" refers to a nucleotide
regulatory
sequence that affects the activity only of DNA sequences on its own molecule
of DNA;
this property usually implies that the locus does not encode for protein.
As used herein, the phrase "trans-acting locus" refers to a nucleotide
regulatory
sequence that affects the activity of a DNA 5' or 3' from its own molecule of
DNA.
As used herein, the term "attenuator" refers to a regulatory nucleotide
sequence
between a promoter and the structural gene of some operons that can act to
regulate the
transit of RNA polymerise and thus control transcription of the structural
gene.
As used herein, the phrase "regulatory non-translatable region sequence"
refers
to a regulatory nucleotide sequence that does not code for a protein or
fragment thereof.
Detectable Labels
As explained above, detectable labels have applications in a variety of
embodiments of the present invention. In particular, numerous labels well
known to
those of ordinary skill in the art can have applications with oligonucleotides
described
herein, as well as at least one molecule of a multidimensional library of the
present
invention. Even a target molecule of a multidimensional library can be
detectably
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labeled using routine techniques. Suitable labels include enzymes,
fluorophores (eg.,
fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR),
rhodamine,
free or chelated lanthanide series salts, especially Eu3+, to name a few
fluorophores),
chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex
particles,
ligands (~., biotin), chemiluminescent agents, enzymes, and amplifiable
nucleotide
sequences. When a control marker is employed, the same or different labels may
be
used for the receptor and control marker.
In the instance where a radioactive label, such as the isotopes 3H, I4C, 3'P,
3sS,
~6C1, SICr, 5'Co, SgCo, 59Fe, 9°Y, lash i3il and IB~Re are used, known
currently available
counting procedures may be utilized. In the instance where the label is an
enzyme,
detection may be accomplished by any of the presently utilized colorimetric,
spectrophotometric, fluorospectrophotometric, amperometric or gasometric
techniques
known in the art.
Direct labels are one example of a label which can be used according to the
present invention. A direct label has been defined as an entity, which in its
natural
state, is readily visible, either to the naked eye, or with the aid of an
optical filter and/or
applied stimulation, e.g., U.V. light to promote fluorescence. Among examples
of
colored labels, which can be used according to the present invention, include
metallic
sol particles, for example, gold sol particles such as those described by
Leuvering (U.S.
Patent 4,313,734); dye sol particles such as described by Gribnau et al. (U.S.
Patent
4,373,932) and May et al. (WO 88/08534); dyed latex such as described by May,
supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in
liposomes as
described by Campbell et al. (U.S. Patent 4,703,017). Other direct labels
include a
radionucleotide, a fluorescent moiety or a luminescent moiety. In addition to
these
direct labeling devices, indirect labels comprising enzymes can also be used
according
to the present invention. Various types of enzyme linked immunoassays are well
known in the art, for example, alkaline phosphatase and horseradish
peroxidase,
lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease,
these
and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay
ELISA and EMIT in Methods iu Efzzymology, 70:419-439(1980) and in U.S. Patent
4,857,453.
Other labels for use in the invention include magnetic beads or magnetic
resonance imaging labels.
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Moreover, molecules of a multidimensional library of the present invention can
also be labeled by metabolic labeling. Metabolic labeling, for example, occurs
during
iya vitro incubation of the cells that express the multidimensional peptides)
in the
presence of culture medium supplemented with a metabolic label, such as [35S]-
methionine or [32P]-orthophosphate. Likewise, isolated oligonucleotides of a
multidimensional library of the present invention can be metabolically labeled
by
replicating in the presence of metabolic labels.
In addition to metabolic (or biosynthetic) labeling with [35S]-methionine, the
invention further contemplates labeling with [1øC]-amino acids and [3H]-amino
acids
(with the tritium substituted at non-labile positions).
Due to the degenerate nature of codons in the genetic code, a multidimensional
peptides) of a multidimensional library of the present invention can be
encoded by
numerous oligonucleotides. "Degenerate nature" refers to the use of different
three-
Ietter codons to specify a particular amino acid pursuant to the genetic code.
It is well
known in the art that a total of 64 codons are known in nature and can be used
interchangeably to code for the twenty naturally occurring amino acid
residues. A list
of the naturally occurring codons are sat forth below:
Phenylalanine (Phe or F) UUU or UUC
Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG
Isoleucine (Ile or I) AUU or AUC or AUA
Methionine (Met or M) AUG
Valine (Va1 or V) GUU or GUC of GUA or GUG
Serine (Ser or S) UCU or UCC or UCA or UCG or AGU
or AGC
Proline (Pro or P) CCU or CCC or CCA or CCG
Threonine (Thr or ACU or ACC or ACA or ACG
T)
Alanine (Ala or A) GCU or GCG or GCA or GCG
Tyrosine (Tyr or Y) UAU or UAC
Histidine (His or H) CAU or CAC
Glutamine (Gln or Q) CAA or CAG
Asparagine (Asn AAU or AAC
or N)
Lysine (Lys or I~) AAA or AAG
Aspartic Acid (Asp GAU or GAC
or D)
Glutamic Acid (Glu GAA or GAG
or E)
Cysteine (Cys or C) UGU or UGC
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Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG
Glycine (Gly or G) GGU or GGC or GGA or GGG
Tryptophan (Trp or W) UGG
Termination codon UAA (ochre) or UAG (amber) or UGA (opal)
5
Methods for synthesizing isolated oligonucleotides that encode for
multidimensional peptides of a multidimensional library of the present
invention
employ 48 of these codons. However, those 48 codons encode for all 20
naturally
occurring amino acid residues. As explained iyzfra, these 48 codons provide
much
10 greater variability to the multidimensional peptides of a library of the
present invention
then do conventional methods of producing peptides for a library. It should be
understood that the codons specified above are for RNA sequences. The
corresponding
codons for DNA have a T substituted for U.
15 Cloning Vectors
Furthermore, the present invention also relates to cloning vectors comprising
oligonucleotides encoding multidimensional peptides of a multidimensional
library of
the invention and an origin of replication, wherein the MDP comprises a
general
formula of (XYn)m, and X is a functional peptide unit, Y is a structural
peptide unit, n is
20 an integer, such that O~n-X10, and m is an integer wherein 2-_err~20. In
another
embodiment, a cloning vector of the present invention comprises at least one
isolated
oligonucleotide of a multidimensional library of the invention, wherein the
isolated
oligonucleotide comprises a general formula of (XY")m, wherein (XYn) is a
repeating
unit of the isolated oligonucleotide, X is a functional unit comprising a
nucleotide
25 regulatory sequence, Y is a structural unit comprising from 6 to at least
60 contiguous
nucleotides, n is the number of the structural units in the repeating unit,
such that
0-~n-X10, and an origin of replication.
A large number of vector-host systems known in the art may be used. Possible
vectors include, but are not limited to, plasmids or modified viruses, but the
vector
30 system must be compatible with the host cell used. Examples of vectors
include, but
are not limited to, E. coli, bacteriophages such as lambda derivatives, or
plasmids such
as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c,
pFLAG, etc. The insertion into a cloning vector can, for example, be
accomplished by
ligating an isolated oligonucleotide into a cloning vector which has
complementary
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36
cohesive termini. However, if the complementary restriction sites used to
fragment the
DNA are not present in the cloning vector, the ends of the DNA molecules may
be
enzymatically modified. Alternatively, any site desired may be produced by
ligating
nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may
comprise specific chemically synthesized oligonucleotides encoding restriction
endonuclease recognition sequences. Isolated oligonucleotides that encode at
least one
multidimensional peptide of a multidimensional library of the invention are
molecules
of a multidimensional library can be introduced into host cells via
transfection,
electroporation, microinjection, transduction, cell fusion, DEAF dextran,
calcium
phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or
a DNA
vector transporter (see, ~., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu
and Wu,
1988, J. Biol. Cl~.em. 263:14621-14624; Hartmut et al., Canadian Patent
Application
No. 2,012,311, filed March 15, 1990), etc., so that many copies of the
oligonucleotides
are generated. The same is true for isolated oligonucleotides that form a
multidimensional library of the present invention, wherein the isolated
oligonucleotide
comprises a general formula of (XYn)m, wherein:
(XYn) is a repeating unit of the oligonucleotide in which:
X is functional unit comprising a nucleotide regulatory sequence,
Y is a structural unit comprising a nucleotide sequence comprising from 6 to
at
least 60 nucleotides,
n is the number of said structural units in said repeating unit, and
m is a number of repeating units in said at least one isolated
oligonucleotide.
Expression Vectors
As explained above, oligonucleotides encoding multidimensional peptides of
libraries of the present invention can be inserted into an appropriate
expression vector,
i.e., a vector which contains the necessary elements for the transcription and
translation
of the inserted protein-coding sequence. Such elements are termed herein a
"promoter." Thus, an isolated oligonucleotide is operatively associated with a
promoter in an expression vector of the invention. An expression vector also
preferably
includes an origin of replication.
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The necessary transcriptional and translational signals can be provided on a
recombinant expression vector, or they may be supplied by a recombinant
oligonucleotide.
Potential host-vector systems include but are not limited to mammalian cell
systems infected with virus (~., vaccinia virus, adenovirus, etc.); insect
cell systems
infected with virus (~., baculovirus); microorganisms such as yeast containing
yeast
vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or
cosmid
DNA. The expression elements of vectors vary in their strengths and
specificities.
Depending on the host-vector system utilized, any one of a number of suitable
transcription and translation elements may be used.
A multidimensional peptide of a multidimensional library of the invention may
be expressed chromosomally, after integration of the coding sequence by
recombination. In this regard, any of a number of amplification systems may be
used
to achieve high levels of stable oligonucleotide expression (See Sambrook et
al., 1989,
supra).
The cells) containing the recombinant vectors) comprising the
oligonucleotide(s) encoding the multidimensional peptides) are cultured in an
appropriate cell culture medium under conditions that provide for expression
of
oligonucleotide(s) by the cell.
Any of the methods previously described for the insertion of DNA fragments
into a cloning vector may be used to construct expression vectors containing
an
oligonucleotide(s) encoding a multidimensional peptides) comprising
appropriate
transcriptional/translational control signals and the protein coding
sequences. These
methods may include in vitro recombinant DNA and synthetic techniques and in
vivo
recombination (genetic recombination).
Expression of an oligonucleotide(s) to produce a multidimensional peptides)
of a multidimensional library of the present invention may be controlled by
any
promoter/enhancer element known in the art, but these regulatory elements must
be
functional in the host selected for expression. Promoters which may be used
include,
but are not limited to, the SV40 early promoter region (Benoist and Chambon,
1981,
Nature 290:304-310), the promoter contained in the 3' long terminal repeat of
Rous
sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the hexpes thymidine
kinase
promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445),
the
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38
regulatory sequences of the metallothionein gene (Brinster et al., 1982,
Nature 296:39-
42); prokaryotic expression vectors such as the (3-lactamase promoter (Villa-
Kamaroff,
et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter
(DeBoer,
et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful
proteins from
recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter
elements
from yeast or other fungi such as the Gal 4 promoter, the ADH (alcohol
dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline
phosphatase promoter; and the animal transcriptional control regions, which
exhibit
tissue specificity and have been utilized in transgenic animals: elastase I
gene control
region which is active in pancreatic acinar cells (Swift et al., 1984, Cell
38:639-646;
Ornitz et al., 1986, Cold Spring Harbor Symp. Quafat. Biol. 50:399-409;
MacDonald,
1987, Hepatology 7:425-515); insulin gene control region which is active in
pancreatic
beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control
region
which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;
Adames et
al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-
1444),
mouse mammary tumor virus control region which is active in testicular,
breast,
lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene
control
region which is active in liver (Pinkert et al., 1987, Gefies and Devel. 1:268-
276),
alpha-fetoprotein gene control region which is active in liver (Krumlauf et
al., 1985,
Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-
antitrypsin gene control region which is active in the liver (Kelsey et al.,
1987, Genes
and Devel. 1:161-171), beta-globin gene control region which is active in
myeloid cells
(Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-
94), myelin
basic protein gene control region which is active in oligodendrocyte cells in
the brain
(Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control
region
which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and
gonadotropic
releasing hormone gene control region which is active in the hypothalamus
(Mason et
al., 1986, Science 234:1372-1378).
Expression vectors comprising at least one isolated oligonucleotide having a
general formula of [(NNB)F~]"" wherein:
NisAorCorGorT/U;
B is C or G or T/LT, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that O~n~lO; and
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39
m is an integer, such that 2-_<m~20,
operatively associated with a promoter, can be identified by four general
approaches:
(a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic
acid
hybridization, (c) presence or absence of selection marker gene functions, and
(d)
expression of inserted sequences. In the first approach, the isolated
oligonucleotides
can be amplified by PCR to provide for detection of the amplified product. In
the
second approach, the presence of a foreign oligonucleotide in an expression
vector can
be detected by nucleic acid hybridization using probes comprising sequences
that are
homologous to an inserted marker gene. In the third approach, the recombinant
vector/host system can be identified and selected based upon the presence or
absence of
certain "selection marker" gene functions (~., ~i-galactosidase activity,
thymidine
kinase activity, resistance to antibiotics, transformation phenotype,
occlusion body
formation in baculovirus, etc.) caused by the insertion of foreign genes in
the vector.
Unicellular Hosts Transformed or Transfected with a Vector of the Present
Inyention
A wide variety of unicellular hosdexpression vector combinations may be
employed in replicating and/or expressing isolated oligonucleotides which form
a
multidimensional library of the present invention, or isolated
oligonucleotides which
encode for multidimensional peptides of multidimensional libraries of the
present
invention. For example, useful expression vectors may consist of segments of
chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors
include derivatives of SV40 and known bacterial plasmids, e.g., E. coli
plasmids col El,
pCRl, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and
their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous
derivatives of
phage ~,, e.g., NM989, and other phage DNA, ~, M13 and filamentous single
stranded phage DNA; yeast plasmids such as the 2~ plasmid or derivatives
thereof;
vectors useful in eukaryotic cells, such as vectors useful in insect or
mammalian cells;
vectors derived from combinations of plasmids and phage DNAs, such as plasmids
that
have been modified to employ phage DNA or other expression control sequences;
and
the like.
For example, in a baculovirus expression systems, both non-fusion transfer
vectors, such as but not limited to pVL941 (BamHl cloning site; Summers),
pVL1393
(BamHl, SnaaI, XbaI, EcoRl, NotI, XmaIII, BgIII, and Pstl cloning site;
Invitrogen),
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pVL1392 (BgIII, PstI, NotI, XmaIII, EcoRI, XbaI, Smal, and BamH1 cloning site;
Summers and Invitrogen), and pBlueBacIl1 (BanzHl, BglII, Pstl, NcoI, and
HindlII
cloning site, with blue/white recombinant screening possible; Invitrogen), and
fusion
transfer vectors, such as but not limited to pAc700 (BamH1 and KpnI cloning
site, in
5 which the BamH1 recognition site begins with the initiation codon; Summers),
pAc701
and pAc702 (same as pAc700, with different reading frames), pAc360 (BamHl
cloning
site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen
(195)), and
pBlueBacHisA, B, C (three different reading frames, with BamHl, BgllI, PstI,
NcoI,
and HindIII cloning site, an N-terminal peptide for ProBond purification, and
10 blue/white recombinant screening of plaques; Invitrogen (220)) can be used.
Mammalian expression vectors contemplated for use in the invention include
vectors with inducible promoters, such as the dihydrofolate reductase (DHFR)
promoter, e.g., any expression vector with a DHFR expression vector, or a
15 DHFRlmethotrexate co-amplification vector, such as pED (PstI, SaII, SbaI,
SnzaI, and
EcoRI cloning site, with the vector expressing both the cloned gene and DHFR;
see
Kaufman, Curz-ezat Protocols in Molecular Biology, 16.12 (1991).
Alternatively, a
glutamine synthetase/methionine sulfoximine co-amplification vector, such as
pEEl4
(HindIIT, XbaI, Smal, SbaI, EcoRI, and BcII cloning site, in which the vector
expresses
20 glutamine synthase and the cloned gene; Celltech). In another embodiment, a
vector
that directs episomal expression under control of Epstein Barr Virus (EBV) can
be
used, such as pREP4 (BamHl, SfiI, XhoI, NotI, NlzeI, HizzdIII, Nh.eI, PvuII,
and KpnI
cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker;
Invitrogen), pCEP4 (BanzHl, S,~ZI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and
Kpf2I
25 cloning site, constitutive hCMV immediate early gene, hygromycin selectable
marker;
Invitrogen), pMEP4 (KpnI, PvuI, NlzeI, HirzdIII, NotI, XlaoI, SfiI, BamH1
cloning site,
inducible metallothionein IIa gene promoter, hygromycin selectable marker:
Invitrogen), pREP8 (BaznHl, XhoI, NotI, HindIII, NheI, and KpnI cloning site,
RSV-
LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NlzeI,
HindIlT,
30 NotI, XlzoI, SfiI, and BamHI cloning site, RSV-LTR promoter, 6418
selectable marker;
Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin selectable marker, N-
terminal peptide purifiable via ProBond resin and cleaved by enterokinase;
Invitrogen).
Selectable mammalian expression vectors for use in the invention include
pRc/CMV
(HizzdIII, BstXI, NotI, SbaI, and ApaI cloning site, 6418 selection;
Invitrogen),
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41
pRc/RSV (Hi»dIII, SpeI, BstXI, NotI, Xbal cloning site, 6418 selection;
Invitrogen),
and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991,
supra)
for use according to the invention include but are not limited to pSCl1 (S»aaI
cloning
site, TK- and [3-gal selection), pMJ601 (SaII, SmaI, AfII, NarI, BspMII,
BamHI, ApaI,
NheI, SacII, Kp»I, and HiradlII cloning site; TK- and (3-gal selection), and
pTKgptFlS
(EcoRI, PstI, SaII, AccI, Hi»dII, SbaI, Ba»aHI, and Hpa cloning site, TK or
XPRT
selection).
Yeast expression systems can also be used in expression vectors of the present
invention as well as to express an oligonucleotide(s) encoding a
multidimensional
peptides) of a library of the present invention. For example, the non-fusion
pYES2
vector (XbaI, SplzI, SIaoI, NotI, GstXI, EcoRI, BstXI, Ba»aHl, SacI, Kp»1, and
Hi»dIl1
cloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI,
BstXI,
EcoRI, BamHl, Sacl, Kp»I, and Hi»dIII cloning site, N-terminal peptide
purified with
ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two,
can be
employed.
Particular examples of vectors having applications herein include
bacteriophage vectors such as 8X174, 1, M13 and its derivatives, fl, fd, Pfl,
etc.,
phagemid vectors, plasmid vectors, insect viruses, such as baculovirus
vectors,
mammalian cell vectors, such as parvovirus vectors, adenovirus vectors,
vaccinia virus
vectors, retrovirus vectors, yeast vectors such as Tyl, killer particles, etc.
Once a suitable host system and growth conditions are established,
recombinant expression vectors can be propagated and prepared in large
quantity. As
previously explained, the expression vectors which can be used include, but
are not
limited to, the following vectors or their derivatives: human or animal
viruses such as
vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast
vectors;
bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to
name
but a few.
In addition, a unicellular host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the gene
product in the
specific fashion desired. Different host cells have characteristic and
specific
mechanisms for the translational and post-translational processing and
modification
(~., glycosylation, cleavage [e.g., of signal sequence]) of proteins.
Appropriate cell
lines or host systems can be chosen to ensure the desired modification and
processing
of the foreign protein expressed. Thus, for example, one can readily modify
the
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42
oligonucleotide(s) that encode multidimensional peptides of a multidimensional
library
to have a signal, sequence instructing the unicellular host to translocate the
multidimensional peptide to the surface of the host. Moreover, other
modifications can
be made to multidimensional peptides, such as glycosylation.
As explained above, vectors are introduced into the desired host cells by
methods known in the art, e.g., transfection, electroporation, microinjection,
transduction, cell fusion, DEAF dextran, calcium phosphate precipitation,
lipofection
(lysosome fusion), use of a gene gun, or a DNA vector transporter (see, ~., Wu
et al.,
1992, J. Biol. Clzezzz. 267:963-967; Wu and Wu, 1988, J. Biol. Cherzz.
263:14621-
14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March
15,
1990).
Commercial Kits
In a further embodiment, the present invention extends to commercial test kits
suitable for use by skilled artisans to produce a multidimensional library
described
herein, and to use such a multidimensional library to screen molecules of a
multidimensional library to determine if any has affinity with a particular
target
molecule. A particular kit of the present invention for screening molecules
that
potentially interact with a target molecule comprises:
(a) a predetermined amount of a multidimensional library (MDL)
comprising at least one molecule that potentially has affinity for the
target molecule, wherein the at least one molecule has a general
formula of (XYn)m, wherein:
X is a functional unit that interacts with the target molecule;
Y is a structural unit;
n is an integer, such that O~n~lO;
m is an integer, such that 2~m~20,
(b) other reagents; and
(c) directions for use of the kit.
Yet another kit for screening molecules that potentially interact with a
target
molecule comprises:
(a) a predetermined amount of a multidimensional library (MDL)
comprising at least one multidimensional peptide that potentially has
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affinity for the target molecule, wherein the at least one
multidimensional molecule has a general formula of (XY")m, wherein:
X is a functional peptide unit that participates in an interaction between
the at least one multidimensional peptide and the target;
Y is a structural peptide unit;
n is an integer, such that O~n~lO;
m is an integer, such that 2-~rr~20,
(b) other reagents; and
(c) directions for use of the kit.
Reagents having applications in such kits are generally those that maintain a
peptide's
native conformation. Examples of such reagents include, but certainly are not
limited
to protease inlubitors, such as PMSF, phosphate buffered saline, TRIS glycine
buffer,
TRIS HCl buffer, etc., wherein the reagents are at physiological pH.
Another class of such a kit employs oligonucleotides. For example, in a
particular embodiment, a kit of the present invention for screening molecules
that
potentially interact with a target molecule comprises:
(a) a predetermined amount of a multidimensional library (MDL)
comprising at least one isolated oligonucleotide that potentially has
affinity for the target molecule, wherein the at least one isolated
oligonucleotide has a general formula of (XYn)m, wherein:
X is a functional unit comprising a nucleotide regulatory sequence that
participates in an interaction between the at least one isolated
oligonucleotide and the target;
Y is a structural unit comprising a nucleotide sequence comprising
from 5 to at least 50 contiguous nucleotides,
n is an integer, such that 0~n~10; and
m is an integer, such that 2~m~20,
(b) other reagents; and
(c) directions for use of the kit.
Particular examples of nucleotide regulatory sequences having applications
herein are discussed above.
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Yet another embodiment of a kit for screening molecules that potentially
interact with a target molecule comprises:
(a) a unicellular host transformed or transfected with an expression vector
comprising at least one isolated oligonucleotide operatively associated with a
promoter,
wherein the at least one isolated oligonucleotide has the general formula of
[(NNB )Fn] "" wherein:
NisAorCorGorT/LT;
B is C or G or T/U, but not A;
F is a codon encoding a predetermined amino acid residue;
n is an integer, such that 0-~n~10; and
m is an integer, such that 2~rn~20;
(b) reagents for expressing the at least one isolated oligonucleotide;
(c) other reagents; and
(d) directions for use the kit.
Examples of reagents having applications in such a kit include those that
promote the maintenance of a protein's native conformation as discussed above,
as well
as reagents that are used to amplify and express oligonucleotides, e.~., PCR
reagents
such as oligonucleotides, oligonucleotide primers, enzymes, gel matrixes,
buffers, etc.
With such kits, skilled artisans can.produce multidimensional library
described
herein, and use it to identify a member of the library that interacts with a
particular
target molecule.
Methods to Identify MDPs and construction of MDL libraries
In an embodiment, the process of the present method for rapidly and
efficiently
identifying novel compounds termed MDPs consists of two steps: (a)
constructing a
library of vectors expressing inserted synthetic oligonucleotide sequences
encoding a
plurality of proteins, polynucleotides and/or peptides as fusion proteins, for
example,
attached to an accessible surface structural protein of a vector; and (b)
screening the
expressed library or plurality of recombinant vectors to isolate those members
producing proteins, polypeptides and/or peptides that bind to a target of
interest. The
nucleic acid sequence of the inserted synthetic oligonucleotides of the
isolated vector is
determined and the amino acid sequence encoded is deduced to identify an MDP
binding domain that binds the target of choice.
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It is, of course, understood that once a library is constructed according to
the
present invention, the library can be screened any number of times with a
number of
different targets of choice to identify MDPs binding the given target. Such
screening
methods are also encompassed within the present invention.
5 A. Synthesis And Assembly Of Oli~onucleotides
In order to prepare a library of vectors expressing a plurality of proteins,
polypeptides and/or peptides MDPs according to the present invention, single
stranded
sets of oligonucleotides are synthesized and assembled in vitro according to
the
following scheme.
10 The synthetic oligonucleotide sequences are designed to encode functional
and
structural peptide units. The functional units are encoded by variant or
unpredicted
oligonucleotides. The structural units are encoded with invariant nucleotide
sequences
of unpredicted length, or they may be encoded with unpredicted nucleotide
sequences
of variant or invariant length comprising structural microlibraries composed
of a
15 limited number of pre-selected amino acids. The size of the structural
peptide units can
also be randomized, and thus affect the overall length multidimensional
peptides of the
library.
Pairs of variant nucleotides in which one individual member is represented by
5'(NNB)n3' and the other member is represented by 3'(NNV)m5' where N is A, C,
G
20 or T; B is G, T or C; V is G, A or C; n is an integer, such that 10 < n <
100, and m is an
integer, such that 10 < m < 100 are synthesized for assembly into synthetic
oligonucleotides. As assembled, according to the present invention, there are
at least
n+m variant codons in each inserted synthesized double stranded
oligonucleotide
sequence.
25 As it would be understood by those skilled in the art, the variant
nucleotide
positions have the potential to encode all 20 naturally occurring amino acids,
whose
naturally occurring codons (64 in total) are described above (non-natural
amino acids
may also be used if an appropriate expression system is used) and, when
assembled as
taught by the present method, encode only one stop eodon, i-e., TAG. The
sequence of
30 amino acids encoded by the variant nucleotides of the present invention is
unpredictable and substantially random in sequence.
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Although the variant oligonucleotides according to the present scheme employ
only 48 different codons to encode all 20 naturally occurring amino acids, the
present
scheme for designing the variant nucleotides advantageously provides greater
variability than that available in conventional schemes, ~. those which use
nucleotides of the formula NNK, in which K is G or T (Cwirla et al., Pf-oc.
Natl. Acad.
Sci. U.S.A. 87: 6378-6382, 1990) or of the formula NNS, in which S is G or C
(Devlin
et al., Scie~ace 249: 404-401, 1990; Scott and Smith, Science 249: 386-390,
1990), in
which only 32 codons are employed.
Moreover, when the synthesized oligonucleotides are inserted into an
expression vector(s), the single stop codon TAG can be suppressed by
expressing the
library of vectors in a mutant host, such as E. coli supE. Other hosts having
applications herein are described above, and in Sambrook, pp. 2.55, 2.57-0.59,
4.13-
4.15.
Moreover, as would be understood by those skilled in the art, use of variant
codons of the formula NNK or NNS would, like the presently employed NNB
formula,
encode only one type of stop codon, i.e., TAG. If the use of suppressors, such
as SupE,
were 100% efficient to suppress the single stop codon, there would be no
difference or
advantage in using the present NNB scheme over those schemes used by
conventional
methods.
The NNB scheme set forth herein offers additional flexibility when the MDPs
are expressed in hosts that lack suppresser tRNA genes. That is, the NNB
scheme
would not be restricted only to host organisms that have been subject to
intense
molecular genetic manipulation and thus offers greater flexibility in host
selection.
One could avoid stop codons altogether by using codon triplets, but then one
would need to know codon preference ideally for each host.
The invariant nucleotides are positioned at particular sites in the nucleotide
sequences to locate variant nucleotides on particular distance form each
other.
The 3' termini invariant nucleotide positions are complementary pairs of 6, 9
or 12 nucleotides to aid in annealing the two synthesized single stranded sets
of
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47
nucleotides together and the conversion to double-stranded DNA, designated
herein-
synthesized double stranded oligonucleotides.
FIG 1B schematically shows the general assembly process according to a
method of the present invention. The oligonucleotides are assembled by a
process
comprising: synthesizing three single stranded nucleotides having a formula
represented:
1) 5'- Complementary site 1 - [(NNB)Fo_n]m - Complementary site 2 - 3' (ON-
69)
2) 3'- Complementary site 1 - 5' (ON-11)
3) 3'- Complementary site 2 - 5' (ON-10)
wherein NNB represents a codon that results in any of the 20 natural amino
acids; F
represents a single pre-synthesized codon, or combination of several single
codons, or
their random pre-synthesized sequences that result in one or combination of
pre-
selected amino acids; n is a number of codons resulting in structural blocks
of amino
acids which is a random value and could be for example, 0-10; m is a number of
functional codons which could be for example, 2-20.
Such random oligonucleotides can be obtained by synthesis in which N
represents equimolar mixture of A, C, G, and T; B represents equimolar mixture
of G,
C, and T.
Any method for synthesis of the single stranded sets of nucleotides is
suitable,
including such as the use of an automatic nucleotide synthesizer. The
synthesizer can
be programmed so that the nucleotides can be incorporated, either in equimolar
or non-
equimolar ratios amounts as the variant positions, i.e., N or B.
In a particular example, a purified single stranded nucleotide sequence,
designated ON-69, is ligated into the SfiI sites of fUSE 5 after annealing to
two "half
site" oligonucleotides, ON-10 and ON-11, which are complementary to the 3' and
5'
portions of ON-69, respectively. "Half site" oligonucleotides anneal to the 5'
and 3'
ends of oligonucleotide ON-69 to form appropriate SfzI cohesive ends. This
will leave
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the appropriate SfiI site exposed without the need to digest with SfiI, thus
avoiding the
cutting of any SfiI site that might have appeared in the variable region.
The scheme for synthesis and assembly of the unpredictable oligonucleotides
used to construct the libraries of the present invention incorporates m
variant,
unpredicted nucleotide sequences of the formula (NNB)m where B is G, T or C
and m
is an integer such that 25m<_20 into the synthesized single stranded
oligonucleotides.
Such a scheme provides a number of important advantages not available with
conventional libraries. As assembled, the present synthesized oligonucleotides
encode
all 20 naturally occurring amino acids by the use of 48 different amino acid
encoding
codons. Thus, the present scheme advantageously provides greater variability
than
other conventional schemes. For example, conventional schemes in which the
variant
nucleotides have the formula NNK, where K is G or T, or NNS, where S is C or
G, use
only 32 different amino acid encoding codons. The use of a larger number of
amino
acid encoding codons may make the present libraries less susceptible to codon
preferences of the host when the libraries are expressed. Although both the
present
scheme and conventional schemes retain only one stop codon, the use of NNB, as
presently taught, advantageously provides synthesized oligonucleotides in
which the
probability of a stop codon is decreased compared to conventional NNS or NNK
schemes.
Additionally, the present scheme avoids the use of synthesized
oligonucleotides rich in GC nucleotides such as often found in libraries using
an NNS
formula for variant codons. As is well known to those skilled in the art,
nucleotide
sequences rich in GC residues are difficult to assemble properly and to
sequence.
Perhaps most significantly, the present scheme for synthesis and assembly of
the oligonucleotides provides sequences of oligonucleotides encoding
unpredicted
amino acid sequences of random length, which are different from any prior
conventional libraries. When constructed according to the present invention,
the
present synthesized single stranded oligonucleotides comprise at least about
27-681
nucleotides in length encoding the complementary site and about 2-20
unpredicted
amino acids (functional units) in the MDP binding domain separated by about 0-
20
structural peptide units of about 0-10 amino acid residues in length.
According to a
particular embodiment, n is 0<n<10 and m is 2<m<20. Thus, the synthesized
single
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stranded oligonucleotides comprise at least 27-687 nucleotides and encode
about 2-20
unpredicted amino acid residues in a functional unit of an MDP. In the
specifically
exemplified examples, the synthesized oligonucleotides encode respectively,
about 4-
16 amino acid residues in the MDP binding domain.
The conventional teaching in the art is that the length of inserted
oligonucleotides should be kept small encoding preferably less than 15 and
most
preferably about 6-8 amino acids and of fixed length. Contrarily, the present
inventors
have found surprisingly and unexpectedly, and in stark contrast to
conventional
teaching, not only can multidimensional libraries encoding products of variant
length
be constructed, but that such libraries can be advantageously screened to
identify
MDPs or proteins, polypeptides and/or proteins having binding specificity for
a variety
of targets.
Among those interested in using computer modeling to identify binding
molecules for drug development, the conventional wisdom has been that the
peptides
used as leads for developing non-peptide mimetics should be kept to a maximum
of
about 6-8 amino acids. Computer modeling of larger peptides has been deemed
impractical or non-informative. Hence, the conventional wisdom has been that
screening libraries of short peptide sequences is more productive. In complete
contrast,
the present invention, which provides methods to efficiently generate and
screen
libraries of peptides that have variant lengths to identify MDPs comprising
functional
peptide units separated to the most optimal distances with structural peptide
elements
that also have the most appropriate flexibility. This can be used later for
drug
development using such computer modeling techniques. Additionally, MDPs
identified by the methods of the present invention afford a whole new vista of
drug
candidates.
As demonstrated in the Examples i~afra, the variable length and the presence
of
structural elements of variable length in oligonucleotides inserted into
expression
vectors affords the ability to identify MDPs wherein a short sequence of amino
acids
split with optimized structural linkers permits the MDPs to possess optimal
specificity
and selectivity for a target with either a simple or complex binding site.
In a particular application, i-e., identification of an MDP having binding
specificity for a large target molecule, multidimensional libraries described
herein
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provide the opportunity to identify or map MDPs that encompass not only a few
contiguous amino acid residues, but also, those that encompass discontinuous
amino
acids.
Additionally, the possibility to optimize positions of functional peptide
units by
5 separating them with structural peptide units in the inserted synthesized
oligonucleotides of multidimensional libraries set forth herein provides an
opportunity
for the development of secondary and/or tertiary structure development in the
potential
MDP, and in sequences flanking the actual functional portions) of the peptide.
Such
complex structural developments are not feasible when only oligonucleotides of
fixed
10 length are used.
B. Insertion Of The Synthetic Oli~onucleotides Into An Appropriate
Exuression Vector
At least one isolated oligonucleotide of appropriate size prepared as
described
above, and particularly, a plurality of such oligonucleotides, is inserted
into an
15 appropriate expression vector. When inserted into a suitable host, this
vector expresses
the plurality of proteins, polypeptides andlor proteins as heterofunctional
fusion
proteins with an expressed component of the vector. These proteins,
polypeptides
and/or proteins are screened to identify MDPs having affinity for a target of
choice.
Any of a variety of vectors can be used according to the methods of the
20 invention, examples of which are described above. Moreover, an appropriate
vector
comprises a gene encoding an effector domain of an MDP to aid expression
and/or
detection of the MDP. At least two different restriction enzyme sites within
such gene,
comprising a linker, are preferred. It is particularly useful to include a
"stuffer
fragment" within the linker region of the vector when the vector (e.~. phage
or
25 plasmid) is intended to express the MDP as a fusion protein that is
expressed on the
surface of the vector. As used in the present application, a "stuffer
fragment" is
intended to encompass a relatively short (i~e., about 14 nucleotides) known
DNA
sequence flanked by at least two restriction enzyme sites, useful for cloning
the DNA
sequences coding for a binding site recognized by a known target, such as an
epitope of
30 a known monoclonal antibody. The restriction enzyme sites at the termini of
the stuffer
fragment are useful for the insertion of the synthesized double stranded
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oligonucleotides, resulting in deletion of the stuffer fragment (Scott and
Smith, Sciezzce
249: 386-390, 1990)
Because of the physical linkage between the expressed heterologous fusion
protein and the phage or plasmid vector containing the stuffer fragment, and
because
the stuffer fragment comprises a known DNA sequence encoding a protein that is
easily detected and immunologically active (i-ee. an immunological marker),
the
presence or absence of the stuffer fragment can be easily detected either at
the
nucleotide level by DNA sequencing, PCR or hybridization, or at the amino acid
level,
i-ee. using an immunological assay. Such determination allows rapid
discrimination
between recombinant (MDP expressing) vectors generated by insertion of the
synthesized double stranded oligonucleotides and non-recombinant vectors.
In one advantageous aspect, the use of a stuffer fragment avoids a problem
often encountered with the use of a conventional polylinker in the vector, i-
ee. the
restriction sites of the polylinker are too close so that adjacent sites
cannot be cleaved
independently and used at the same time.
In a particular embodiment, the vector is, or is derived from, a filamentous
bacteriophage, including but not limited to M13, fl, fd, Pfl, etc., vector
encoding a
phage structural protein, preferably a phage coat protein, such as pIII,
pVIII, etc.
Moreover, the filamentous phage is an fd derived phage vector such as fUSES
described in Scott and Smith (Science 249: 386-390, 1990) which encodes the
structural coat protein pIII. Other vectors having applications in the present
invention
are described above.
The phage vector is chosen to contain, or is constructed to contain, an origin
of
replication located in the S' region of a gene encoding a bacteriophage
structural protein
so that the plurality of the synthesized oligonucleotides inserted are
expressed as fusion
proteins on the surface of the bacteriophage. This advantageously provides not
only a
plurality of accessible expressed proteins/peptides but also provides a
physical link
between the proteins/peptides and the inserted oligonucleotides to provide for
easy
screening and sequencing of the identified MDPs.
In addition, according to a particular embodiment, the structural
bacteriophage
protein is pIII; The fCTSES vector described by Smith et al., and illustrated
in FIG 1A,
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containing the pIII gene having a 14-by "stuffer fragment" introduced at the N-
terminal
end, flanked by two SfiI restriction sites was used in examples exemplified in
Section
6. The library is constructed by cloning the plurality of synthesized
oligonucleotides
into a cloning site near the N-terminus of the mature coast protein of the
appropriate
vector, preferably the pIII protein, so that the oligonucleotides are
expressed as coast
protein-fusion proteins.
C. Expression Of Vectors In Appropriate Hosts
As explained above, once the appropriate expression vectors are prepared, they
are inserted into an appropriate host or used in transcription and translation
system in
vitro. Methods of transfecting and transforming unicellular hosts are
described above.
The oligonucleotides are expressed by culturing the transformed or transfected
unicellular hosts under appropriate culture conditions for colony or phage
production.
Preferably, the host cells are protease deficient and may or may not carry
suppresser
tRNA genes.
For example, a small aliquot of the electroporated cells is plated and the
number of colonies or plaques is counted in order to determine the number of
recombinants. The library of recombinant vectors in host cells is plated at
high density
for a single amplification of the recombinant vectors.
Moreover, in a particular embodiment of the invention, recombinant fd vector
fUSE5, engineered to contain the synthesized double stranded oligonucleotides
according to the invention, are transfected into MC1061 E. coli cells by
electroporation. MDPs are expressed on the outer surface of the viral capsid
extruded
from the host E. coli cells are accessible for screening. The parent fUSE 5
vector
contains the 14-by stuffer fragment. When the double stranded synthesized
oligonucleotides are inserted between the two SfiI sites, the stuffer fragment
is
removed.
Optionally, several different strains of E. coli may be electroporated to
establish different versions of the same library. Of course, the same E. coli
strain
would need to be used for the entire set of screening experiments. This
strategy is
based on the consideration that there is likely an in vivo biological
selection, both
positive and negative, on the viral assembly, secretion, and infectivity rate
of individual
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' S3
fd recombinants due to the sequence nature of the peptide-pITI fusion
proteins.
Therefore, E. coli yvith different genotypes (i-e., chaperone over-expressing,
or
secretion enhanced) will serve as bacterial hosts, because they will yield
libraries that
differ in subtle, unpredictable ways.
D. Methods To Identify MDPs: Screening Of MDL Libraries
Once a multidimensional library of the present invention is available, it can
be
screened to identify molecules) of the library that interact with a target of
choice. As
stated above, in the present invention, a target is intended to encompass a
substance,
including a molecular complex, a molecule or portion thereof, for which a
protein
receptor naturally exists or can be prepared according to the method of the
invention.
Thus in the present invention, a target is a substance that specifically
interacts with the
functional elements of an MDP and includes, but is not limited to, a chemical
group, an
ion, a metal, a protein, a glycoprotein or any portion thereof, a peptide or
any portion of
a peptide, a nucleic acid or any portion of a nucleic acid, a sugar, a
carbohydrate or a
carbohydrate polymer, a lipid, a fatty acid, a vital particle or portion
thereof, a
membrane vesicle or portion thereof, a cell wall component, a synthetic
organic
compound, a bio-organic compound and an inorganic compound.
Screening the MDL libraries of the present invention can be accomplished by
any of a variety of methods known to those of skill in the art.
If the MDPs are expressed as fusion proteins with a cell surface molecule,
then
screening is advantageously achieved by incubating the vectors with an
immobilized
target and harvesting those vectors that bind to the target. Such useful
screening
methods designated "panning" techniques are described in Parmley et al., (Gene
73:
305-318, 1988). In panning methods useful to screen the present libraries, the
target
can be immobilized on plates, beads, such as magnetic beads, sepharose beads
used in
columns, etc. In particular embodiments, the immobilized target can be
"tagged", i-e.,
using such as biotin, fluorochrome, etc., for FACS sorting.
In a particular embodiment, screening a library of phage expressing MDPs, i-
ee.
phage and phagemid vectors was achieved as follows: using microtiter plates,
the target
was first diluted, i.e. in 100 mM NaHC03, pH 8.5 and a small aliquot of target
solution
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was adsorbed onto wells of microtiter plates (by incubation overnight at
4° C). An
aliquot of BSA solution (1 mglml, in 100 mM NaHC03, pH 8.5) was added and the
plate incubated at room temperature for 1 hr. The contents of the microtiter
plate were
flicked out and the wells washed carefully with PBS-0.5% Tween 20. The plates
were
washed free of unbound targets repeatedly. A small aliquot of phage solution
was
introduced into each well and the wells are incubated at room temperature for
1-2 hrs.
The contents of the microtiter plates were flicked out and washed repeatedly.
The
plates were incubated with wash solution in each well for 10 min at room
temperature
to allow bound phages with rapid dissociation constants to be released. The
wells were
then washed five more times to remove all unbound phages.
In order to recover the phage bound to the wells, a pH change was used. An
aliquot of 50 mM glycine-HCl (pH 2.2), 100 mg/ml BSA solution was then added
to
the washed wells to denature the proteins and release the bound phages. After
10 nnin,
the contents were transferred into clean tubes and a small aliquot of 1 M Tris-
HCl (pH
7.5) or 1 M NaH2P04 (pH 7.0) was added to neutralize the pH of the phage
sample.
The phages were then diluted, ~, 10-3-10-~ and aliquots plated with E. coli
K9lKan
cells to determine the number of the plaque forming units of the sample. The
titer of
the input samples was also determined for comparison (dilutions are generally
10-6-10-
3)'
An important aspect of screening the libraries is the elution. For clarity of
explanation, the following is discussed in terms of MDP expression by phages;
however, it is readily understood that such discussion is applicable to any
system where
the MDP is expressed on a surface fusion molecule. It is conceivable that from
a
plurality of proteins expressed on phages, the conditions that disrupt the
peptide-target
interactions during recovery of the phages are specific for every given
peptide
sequence. For example, certain interactions may be disrupted by acid pHs but
not by
basic pHs, and vice versa. Thus, it is important to test a variety of elution
conditions
(including, but not limited to, pH 2-3, pH 12-13, excess target in
competition,
detergents, mild protein denaturants, urea, varying temperature, light,
presence or
absence of metal ions, chelators, etc.) and compare the primary structures of
the MDP
proteins expressed on the phages recovered for each set of conditions in order
to
determine the appropriate elution conditions for each target/MDP combination.
Some
of these elution conditions may be incompatible with phage infection because
they are
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bactericidal and will need to be removed by dialysis i.e. dialysis bag,
Centricon/Amicon microconcentrators).
The ability of the diffexent multidimensional peptides to be eluted under
different conditions may not only be due to the denaturation of the specific
peptide
5 region involved in binding to the target but may also be due to
conformational changes
in the flanking regions. These flanking sequences may also be denatured in
combination with the actual binding sequence; these flanking regions may also
change
their secondary or tertiary structure in response to exposure to the elution
conditions
(i-ee. pH 2-3, pH 12-13, excess target in competition, detergents, mild
protein
10 denaturants, urea, heat, cold, light, metal ions, chelators, etc.) which in
turn leads to the
conformational deformation of the peptide responsible for binding to the
target.
E. Applications And Uses Of MDPs And MDP Compositions
The MDP products can be used in any industrial or pharmaceutical application
15 that uses a peptide binding moiety specific for any given target. The MDPs
can also be
intermediates in the production of unifunctional binding peptides that are
produced and
selected by the method of the invention to have a binding affinity,
specificity and
avidity for a given target. Thus, according to the present invention, MDPs and
MDP
compositions are used in a wide variety of applications including, but not
limited to
20 uses in the field of biomedicine; biologic control and pest regulation;
agriculture;
cosmetics; environmental control and waste management; chemistry; catalysis;
nutrition and food industries; military uses; climate control;
pharmaceuticals; etc.
The MDPs and MDP compositions are also useful in a wide variety of in vivo
applications in the fields of biomedicine, bioregulation, and control. In
certain
25 applications, the MDPs are employed as mimetic replacements for
compositions such
as enzymes, hormone receptors, immunoglobulins, metal binding proteins,
calcium
binding proteins, nucleotide binding proteins, adhesive proteins such as
integrins,
adhesins, lectins, etc. In other applications, the MDPs are employed as
mimetic
replacements of pxotein/peptides, sugars or other molecules that bind to
receptor
30 molecules, such as for example, mimetics for molecules that bind to
streptavidin,
immunoglobulins, cellular receptors, etc.
Other in vivo uses include administration of MDPs and MDP compositions as
immunogens for vaccines, useful for active immunization procedures. MDPs can
also
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be used to develop immunogens for vaccines by generating a first series of
MDPs
specific for a given cellular or viral macromolecule target and then
developing a second
series of MDPs that bind to the first MDPs, i.e. the first MDP is used as a
target to
identify the second series of MDPs. The second series of MDPs will mimic the
initial
cellular or viral macromolecular target site but will contain only relevant
peptide
binding sequences, eliminating irrelevant peptide sequences. Either the entire
MDP
developed in the second series, or the binding domain, or a portion thereof,
can be used
as an immunogen for an active vaccination program.
In irz vivo applications, MDPs and MDP compositions can be administered to
animals and/or humans by a number of routes including injection (i-ee,
intravenous,
intraperitoneal, intramuscular, subcutaneous, intraarticular, intramammary,
intraurethrally, etc.), topical application, or by absorption through
epithelial or
mucocutaneous linings. Delivery to plants, insects and protists for bio-
regulation
and/or control can be achieved by direct application to the organism,
dispersion in the
habitat, addition to the surrounding environment or surrounding water, etc.
Moreover, in the chemical industry, MDPs can be employed for ,use in
separations, purifications, preparative methods, catalysis, etc.
In addition, MDPs can also be used in the field of diagnostics to detect
targets
occurring in lymph, blood, feces, saliva, sweat, tears, mucus, or any other
physiological
liquid or solid. In the area of histology and pathology, MDPs can be used to
detect
targets in tissue sections, organ sections, smears, or in other specimens
examined
macroscopically or microscopically. MDPs can also be used in other diagnostics
as
replacements for antibodies, as for example in hormone detection kits, or in
pathogen
detection kits, etc., where a pathogen can be any pathogen including bacteria,
viruses,
mycoplasma, fungi, protozoan, etc. MDPs may also be used to define the
epitopes that
monoclonal antibodies bind to by using monoclonal antibodies as targets for
MDP
bindings, thereby providing a method to define the epitope of the original
immunogen
used to develop the monoclonal antibody. MDPs or the binding domain or a
portion
thereof can thus serve as epitope mimetics and/or mimotopes.
Other applications will be readily apparent to those of skill in the art and
are
intended to be encompassed by the present invention.
The present invention may be better understood by reference to the following
non-limiting Examples that are provided as exemplary of the invention. The
following
Examples are presented in order to more fully illustrate the preferred
embodiments of
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57
the invention. They should in no way be construed, however, as limiting the
broad
scope of the invention.
EXAMPLES
The description of the methods for the construction of MDL can be further
subdivided into; (1) synthesis and assembly of synthetic oligonucleotides; (2)
insertion
of the synthetic nucleotides into an appropriate expression vector; and (3)
expression of
the MDL library in vectors.
Reagents And Strains Used In The Examines
SfiI and BgII restriction endonucleases, T4 DNA ligase, T4 kinase, Klenow
polymerise were obtained from Boehringer Mannheim. Sequenase T7 was obtained
from Pharmacia. Oligonucleotides were synthesized with an applied Biosystems
PCR-
Mate Synthesizer and purified on ODC columns (ABI). The fUSE 5 vector and E.
coli
MC1061, K802, K9lKan were kindly provided by Professor George Smith,
University
of Missouri, Columbia, MO and described in Smith et al., (Scieyzce 228: 1315-
1317,
1985) and Parmley and Smith, (Geyae 73: 305-318, 1988).
Example 1:
~nthesis And Assembly Of Oli~onucleotides
FIG. 1B shows the formula of the oligonucleotides and the assembly scheme
used in the construction of the MDL. The oligonucleotides were synthesized
with an
applied Biosystems PCR-Mate synthesizer. The 5'- and 3'- ends have a fixed
sequence, chosen to reconstruct the amino acid sequence in the vicinity of the
signal
peptidase site. The central portion contained the variable regions that
comprise the
oligonucletide library members, and may also code for spacer residues on
either or both
sides of the variable sequence.
This sequence, designated ON-69, was ligated into the S,fiI sites of fUSE 5
after
annealing to the two "Half-site" oligonucleotides, ON-10 (5'-AAGCGCCACC-3')
(SEQ. m. NO.: 1) and ON-11 (5'-ACCGGCCCCGT-3') (SEQ. 1D. N0.:2), which are
complementary to the 3'- and 5'- portions of ON-69, respectively. "Half-site"
oligonucleotides anneal to the 5'- and 3'- ends of oligonucleotide ON-69 to
form
appropriate SCI cohesive ends. This left the appropriate SfiI site exposed
without the
need for digestion with SfiI, thus avoiding the cutting of any SfcI sites that
might have
appeared in the variable region. Oligonucleotides were phosphorylated with T4
kinase
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and annealed in 20 mM Tris-HC1, pH 7.5, 2 mM MgCl2, 50 mM NaCI by mixing 4 ~g
of ON-IO and 4 ~g of ON-I 1 with 2.75 ~ g of ON-69, heating to 65°C for
5 nnin and
allowing to cool slowly to room temperature. This represented an approximate
molar
ratio of 1:10:10 (ON-69: ON-10: ON-11).
Example 2.
Strategy Of The ~~Snlit-Pull" Synthesis
Another way to produce the random polynucleotide is to use successive
splitting and uniting steps ("split-pull" synthesis) during the synthesis as
schematically
shown in FIG 2. The oligonucleotide was synthesized with the starting linker
sequence
5'-GGGCCGGT-N1N2N3- (SEQ. 1D. N0.:3) on a resin support, where Nl is A, C, G
and T (nominally equimolar); NZ is A (31%), C (19%), G (19%), and T (31%); N3
is C
(39%), G (39%), and T (22%). The resin support was then divided into four
fractions
and synthesis continued in each fraction separately according to the following
scheme:
Part 1 (30%): Resin-GGGCCGGT-N1NZN3- (SEQ. ff~. N0.:3)
Part 2 (17%): Resin-GGGCCGGT-N1NZN3-GGT- (SEQ.1D. N0.:4)
Part 3 (23%): Resin-GGGCCGGT-N1N2N3-(GGT)2- (SEQ. ID.
N0.:5)
Part 4 (30%): Resin-GGGCCGGT-N1NZN3-(GGT)3- (SEQ. 1D.
N0.:6)
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All resin particles were thoroughly mixed together and synthesis continued by
adding random -N1NZN3- sequence resulting in Resin-GGGCCGGT-N1NZN3-(GGT)3-
N1N2N3 (SEQ. ID. N0.:7). This protocol was then repeated four times; after
which the
closing linker sequence -GGTGGCGCTTCTG-3' (SEQ. ID. N0.:8) was added. The
final mixture was detached from the resin. The stochastic collection of
polynucleotides
of the general formula 5'-GGGCCGGT{N1NZN3(GGT)°_3}N1NZN3
GGTGGCGCTTCTG-3' was thus obtained. This sequence can be ligated into the SfiI
sites of fCTSE 5 after annealing to two "half site" oligonucleotides, ON-10
(5'-
AAGCGCCACC-3') (SEQ. ID. N0.:1) and ON-11 (5'-ACCGGCCCCGT-3') (SEQ.
~. N0.:2), which are complementary to the 3'- and 5'- portions of the
sequence,
respectively.
Example 3.
Construction Of The MDL Library
The vector fUSE 5 (100 fig) was digested to completion with Sfd and ethanol
precipitated twice in the presence of 2 M ammonium acetate. This DNA could not
be
self-ligated, indicating complete removal of the 14-by "stuffer" that lies
between the
SfiI sites (Figure 1A). Twenty ~g of SfiI digest of fUSE 5 vector was then
ligated with
200 ng of annealed oligonucleotide insert (molar ratio 1:5) by an overnight
incubation
at 15° C in 1 m1 of T4 Iigase buffer (20 mM Tris-HCI, pH 7.5, 5 mM
MgCl2, 2 mM
DTT, 1 mM ATP) and 4000 units of T4 DNA Iigase. The Iigated DNA was ethanol
precipitated in the presence of 0.3 M sodium acetate, xesuspended in 40 p.1 of
water,
and transformed by electroporation into E. coli MC1061. Ten electro-
transformations,
each containing 80 p1 of cell suspensions (final concentration 5x101°
cells/mI) and 2 pg
of DNA (500 pg/m1), were performed by pulsing at 12.5 kV/cm for 5 msec as
described in Dower et al., (Nucleic Acids Res. 16:6127-6145 (1988). After
electroporation, E. coli. cells were allowed to undergo non-selective
outgrowth at 37° C
for 1 hr in 2 ml of SOC medium (consisting of 2% Bacto tryptone, 0.5% Bacto
yeast
extract, 10 mM NaCI, 2.5 mM KC, 10 mM MgCl2, 10 mM MgSOd, 20 mM glucose; as
described by Hanahan et al., (J. Mol. Biol. 166:557-580 (1983)) containing 0.2
mg/ml
tetracycline. Aliquots (20 ~tl) of cells from each of the transformants were
then
removed and various dilutions plated on LB plates (Lucia-Bertani medium)
containing
mg/ml tetracycline to assess the transformation efficiency. The remainder of
the
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cell suspension was used to inoculate 1L of L-broth containing tetracycline
(20 mg/ml)
and was grown through approximately 10 doublings at 37° C to amplify
the library.
Phages from liquid cultures were obtained by clearing the supernatant twice by
centrifugation (8000 RPM for 10 min at 4° C), precipitation of phage
particles with
5 polyethylene glycol (final concentration 3.3% polyethylene glycol-8000, 0.4
M NaCI),
and centrifugation as described above. Phage pellets were re-suspended in TBS
(50
mM Tris-HCI, pH 7.5, 150 mM NaCI) and stored at 4° C. A portion of the
library was
used to infect I~9lKan cells that were plated at low density on LB
tetracycline plates
(40 mg/ml).
Example 4.
Characterization Of The MDL
Constructing a library of peptides displayed on the N-terminus of processed
pIII necessarily alters the amino acids in the vicinity of the signal
peptidase cleavage
site. Certain changes in the corresponding region of the major coat protein,
pVIII, have
been shown to reduce processing efficiency, slowing or preventing the
incorporation of
pVIII to virions (Felici et al. J. Mol. Biol. 222: 301-310, 1991). If all the
pIII were
similarly affected, the diversity of peptides contained in the library would
be reduced
(Parmley and Smith, Gehe 73: 305-313, 1988). The finding that most amino acids
appear at each position of the variable peptides of randomly chosen phage
indicates
that processing defects do not impose important constraints on the diversity
of the
library. Furthermore, it is indicative that the inserted sequence in the
fusion protein
does not deleteriously alter the biological properties of the bacteriophage
protein.
In order to determine whether any coding bias existed in the variant non
predicted peptides expressed by these libraries, perhaps due to biases imposed
during ifa
vitro synthesis of the oligonucleotides, or irc vivo during the expression by
the
reproducing phages, inserted synthetic oligonucleotide fragments of 20
randomly
chosen isolates were examined from the MDP library. Individual clones
producing
infectious phages were picked, and the DNA of their variable region was
sequenced
using sequenase T7 kit and an oligonucleotide sequencing primer fUSE32P (5'-
TGAATTTTCTGTATGAGG-3') (SEQ. 1D. N0.:9), which is complementary to the
sequence located 32 nucleotides to the 3' side of the second Sfil site in the
fUSE 5
vector.
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In FIG. 3, the amino acid frequencies are deduced for the peptides encoded by
the oligonucleotide inserts of a sample of randomly chosen infectious phage.
It is
observed that very few (<10%) of the inserted oligonucleotide sequences
characterized
so far in the library have exhibited complete deletions. The percentage of the
various
deletions is equal to 65%. This is likely a reflection of the heavy G content
in the
structural peptide units) coding part in assembling the oligonucleotides. FIG.
4 shows
distribution of amino acids in the library. Microsoft EXCEL program was used
to
evaluate amino acid frequencies. Such analyses showed that the nucleotide
codons
coding fox, and hence most amino acids, occurred at the expected frequency in
the
MDP library of expressed proteins. The notable exceptions were leucine and
serine,
which were over-represented (FIG. 4A). Thus, except for the structural block
composition limited to 5 amino acids, any position in the variable domain
could have
any amino acid. Therefore, the sequences are unpredicted or random. In the
structural
peptide units all the five amino acids are distributed within two-fold margin
of
theoretical distribution that is equal 20% (FIG. 4B). The structural peptide
units have a
length between 1 and 3 amino acids, and are distributed between 20% and 50%
(FIG.
4C).
Example 5.
Identification Of Target Binding MDPs
Streptavidin was diluted to 200 pg/ml in 0.1 M NaHC03, and 50 ~1 of the
solution was added to each well and used to select clones from MDL by
successive
rounds of biopanning on 96-well plates (Nunc maxisorb microtiter plate).
Streptavidin
was then bound to the plate overnight at 4° C. The wells were then
washed with PBS
and blocked with 1% BSA in 0.1 M NaHC03 for 1 hr at room temperature. After
blocking, the wells were washed six times with 0.1% Tween20/TBS (T-TBS). The
2x1011 phage particles/well of the primary MDL were then added in 100 ~1 of
0.1%
BSA/T-TBS and the plates were incubated for 2 hrs at room temperature. The
plates
were then washed 12 times with T-TBS to remove non-specific phages (phages
which
express peptides without the desired specificity) and the remaining bound
phages were
eluted by a 10-min treatment with 100 ~.l of 0.1 M HCl (pH 2.2 adjusted with
glycine).
Neutralization of the eluate, titration, and amplifications on agar medium
were carried
out essentially as described in Parmley and Smith, (Gene 73:305-313 (1988)).
The
binding and elution reactions were repeated five times. Recoveries of phages
from this
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62
process are shown in FIG. 5, where the repeated selection of phages resulted
in an
enrichment of phages capable of binding to streptavidin. These results
indicate that
phages of higher affinity were preferentially enriched in each panning step.
After five rounds of biopanning and phage amplification, the individual phages
derived from second, third, and fourth rounds of panning were grown and their
peptide
encoding regions sequenced. The amino acid sequences of these 29 phages that
bound
to streptavidin are summarized in FIG. 6.
CONCLUSION
The results of these Examples readily demonstrate that methods of the present
invention set forth herein provide a novel and useful multidimensional library
comprising multidimensional peptides that vary in size, and are not limited to
a
particular size. Thus, libraries described herein permit exploration of the
effect of
secondary and tertiary structure of polypeptides on the ability of proteins
and
polypeptides to interact with, and particularly bind with a target molecule.
In addition,
since multidimensional peptides of libraries described herein comprise both
functional
and structural peptide units, the potential affinity of a multidimensional
peptide can be
maximized, thus ensuring an accurate model of a protein that interacts with
the target.
Furthermore, a novel and useful method of producing oligonucleotides that
encode the
multidimensional peptides of a multidimensional library, as set forth herein
results in
multidimensional peptides that have a limited amount of stop codons, have
random
amino acid sequences, and do not have a maximum length. As a result, the
number of
multidimensional peptides, and thus the number of members of the library
available to
interact with the target is maximized.
The present invention is not to be limited in scope by the specific
embodiments
describe herein. Indeed, various modifications of the invention in addition to
those
described herein will become apparent to those skilled in the art from the
foregoing
description and the accompanying figures. Such modifications are intended to
fall
within the scope of the appended claims.
It is further to be understood that all base sizes or amino acid sizes, and
alI
molecular weight or molecular mass values, given for nucleic acids or
polypeptides are
approximate, and are provided for description.
Various publications are cited herein, the disclosures of which are
incorporated
by reference in their entireties.
