Note: Descriptions are shown in the official language in which they were submitted.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
DNA LIBRARY
Field of the Invention
The present invention relates to a library of DNA sequences immobilised onto a
solid support
and its use in methods of selecting and designing polypeptides comprising
nucleic acid
binding motifs, in particular zinc finger polypeptides.
Background of the Invention
Selective gene expression is mediated via the interaction of protein
transcription factors with
specific r..:cleotide sequences within the regulatory region of the gene. The
most widely used
domain within protein transcription factors appears to be the zinc finger (Zf)
motif. This is an
independently folded zinc-containing mini-domain which is used in a modular
repeating
fashion to achieve sequence-specific recognition of DNA. The first zinc finger
motif was
identified in the Xenopus transcription factor TFIIIA. The structure of Zf
proteins has been
determined by NMR studies (Lee et al., 1989 Science 245, 635-637) and
crystallography
(Pavletich & Pabo, 1991, Science 252, 809-812).
The manner in which DNA-binding protein domains are able to discriminate
between
different DNA sequences is an important question in understanding crucial
processes such as
the control of gene expression in differentiation and development. The zinc
finger motif has
been studied extensively, with a view to providing some insight into this
problem, owing to
its remarkable prevalence in the eukaryotic genome, and its important role in
proteins which
control gene expression in Drosophila, mice and humans (Kinzler et al., 1988
Nature
(London) 332, 371).
Most sequence-specific DNA-binding proteins bind to the DNA double helix by
inserting an
a-helix into the major groove. Sequence specificity results from the
geometrical and
chemical complementarity between the amino acid side chains of the and the
accessible
groups exposed on the edges of base-pairs. In addition to this direct reading
of the DNA
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
sequence, interactions with the DNA backbone stabilise the complex and are
sensitive to the
conformation of the nucleic acid, which in turn depends on the base. ,4
priori, a simple set of
rules might suffice to explain the specific association of protein and DNA in
all completes,
based on the possibility that certain amino acid side chains have preferences
for particular
base-pairs. However, crystal structures of protein-DNA complexes have shown
that proteins
can be idiosyncratic in their mode of DNA recognition, at least partly because
they may use
alternative geometries to present their sensory a-helices to DNA, allowing a
variety of
different base contacts to be made by a single amino acid and vice versa
(Matthew's 1988
Nature (London) 33~, 294-29~).
Mutagenesis of Zf proteins has confirmed modularity of the domains. Site
directed
mutagenesis has been used to change key Zf residues, identified through
sequence homology
alignment, and from the structural data, resulting in altered specificity of
Zf domain (Nardelli
et al., 1992 NAR 26, 4137-4144). The authors suggested that although design of
novel
1 ~ binding specificities would be desirable, design would need to take into
account sequence and
structural data. They state "there is no prospect of achieving a zinc finger
recognition code".
Despite this, many groups have been trying to work towards such a code,
although only
limited rules have so far been proposed. For example, Desjarlais et al.,
(1992b PNAS 89.
734-7349) used systematic mutation of two of the three contact residues (based
on
consensus sequences) in finger two of the polypeptide Spl to suggest that a
limited
degenerate code might exist. Subsequently the authors used this to design
three Zf proteins
with different binding specificities and affinities (Desjarlais & Berg, 1993
PNAS 90, 22~0
2260). They state that the design of Zf proteins with predictable
specificities and affinities
2~ ''may not always be straightforward".
The crystal structures of zinc finger-DNA complexes show a semiconserved
pattern of
interactions in which 3 amino acids from the a-helix contact 3 adjacent bases
(a triplet) in
DNA (Pavletich & Pabo 1991 Science 2~2, 809-817; Fairall et al., 1993 Nature
(London)
366, 483-487; and Pavletich & Pabo 1993 Science 261, 1701-1707). Thus the mode
of DNA
recognition is principally a one-to-one interaction between amino acids and
bases. Because
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
_,_
zinc fingers function as independent modules, it should be possible for
fingers with different
triplet speciticities to be combined to give specific recognition of longer
DNA sequences.
Each finger is folded so that three amino acids are presented for binding to
the DNA target
sequence, although binding may be directly through only rivo of these
positions. In the case
of Zif?68 for example, the protein is made up of three fingers which contact a
9 base pair
contiguous sequence of target DNA. A linker sequence is found between fingers
which
appears to make no direct contact with the nucleic acid.
Protein engineering experiments have shown that it is possible to alter
rationally the
DNA-binding characteristics of individual zinc fingers when one or more of the
a-helical
positions is varied in a number of proteins (Nardelli et al., 1991, Nature
(London) 349,
17~-178; Nardelli et al., 1992, Nucleic Acids Res. 20, 4137-4144: and
Desjarlais & Berg
1992a, Proteins 13, 272). It has already been possible to propose some
principles relating
amino acids on the a-helix to corresponding bases in the bound DNA sequence
(Desjarlais &
1 ~ Berg 1992b, Proc. Natl. Acad. Sci. USA 89, 734-7349). However in this
approach the
altered positions on the a-helix are prejudged, making it possible to overlook
the role of
positions which are not currently considered important; and secondly. owing to
the
importance of context, concomitant alterations are sometimes required to
affect specificity
(Desjarlais & Berg 1992b), so that a significant correlation between an amino
acid and base
may be misconstrued.
To investigate binding of mutant Zf proteins, Thiesen and Bach (1991 FEBS 283,
23-26)
mutated Zf fingers and studied their binding to randomised oligonucleotides,
using
electrophoretic mobility shift assays. Subsequent use of phage display
technology has
2~ permitted the expression of random libraries of Zf mutant proteins on the
surface of
bacteriophage. The three Zf domains of Zif268, with 4 positions within finger
one
randomised, have been displayed on the surface of filamentous phage by Rebar
and Pabo
(1994 Science 263, 671-673). The library was then subjected to rounds of
affinity selection
by binding to target DNA oligonucleotide sequences to obtain Zf proteins with
new binding
specificities. Randomised mutagenesis (at the same postions as those selected
by Rebar &
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-4-
Pabo) of finger 1 of Zif 268 with phage display has also been used by Jamieson
et al., (1994
Biochemistn,~ 33, 5689-5690 to create novel binding specificity and affinity.
More recently Wu et al. (199 Proc. Natl. Acad. Sci. USA 92, 34-1-348) have
made three
libraries, each of a different finger from Zif268, and each having sic or
seven a-helical
positions randomised. Six triplets were used in selections but did not return
fingers with any
sequence biases; and when the three triplets of the Zif268 binding site were
individually used
as controls, the vast majority of selected fingers did not resemble the
sequences of the wild-
type Zif268 fingers and, though capable of tight binding to their target sites
in vitro, were
usually not able to discriminate strongly against different triplets. The
authors interpret the
results as evidence against the existence of a code.
In summary, it is known that Zf protein motifs are widespread in DNA binding
proteins and
that binding is via three key amino acids, each one contacting a single base
pair in the target
1 ~ DNA sequence. Motifs are modular and may be linked together to form a set
of fingers
which recognise a contiguous DNA sequence (e.g. a three fingered protein will
recognise a
9mer etc). The key residues involved in DNA binding have been identified
through sequence
data and from structural information. Directed and random mutagenesis has
confirmed the
role of these amino acids in determining specificity and affinity. Phage
display has been used
to screen for new binding specificities of random mutants of fingers. A
recognition code, to
aid design of new finger specificities, has been worked towards although it
has been
suggested that specificity may be difficult to predict.
Given the lack of predictability in the outcome of rational zinc finger
engineering. there is a
2~ need for a reliable method for checking the results of efforts to custom
design zinc fingers
with desired sequence specificity, whether such zinc fingers are obtained by
design ("rational
design") or by selection from random mutants (''empirical selection''). Not
Qnly should the
target sequence be included in the test assay but also related sequences
because~(i) selection is
by affinity and not necessarily by specificity and (ii) as discussed, rational
design is unreliable
owing to degenerate recognition codes, incomplete code and/or unpredictable
synergistic
contacts.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-5-
Ideally, the assay should include all possible DNA sequences, of given length,
to establish
the preferred specificity of the protein motif to rank other acceptable DNA
sequences in terms
of affinity. Therefore, wherever possible, an idea of the absolute affinity
should emerge in
parallel, i.e. the assay should not be simply comparative. This is possible
by, for example,
determining the apparent Kd of a protein for a series of related binding
sites.
However, as the number of test binding sites in the assay increases, it
becomes unfeasible to
achieve this using prior art techniques. One possible method is to use the
SELEX technique
(Thiesen and Bach, 1991, FEBS 283, 23-26). However this technique is (i)
iterative and
hence laborious, (ii) comparative not quantitative, no Kds emerge, (iii)
requires empirical
determination of starting parameters and (iv) if selection rounds are earned
too far then all
comparative information is lost too, as only the best site survives the
selection. In addition.
as selection is exponential (by PCR) very small differences in DNA-binding
preferences can
1 ~ result in apparently huge selection pressures.
Smmmarv of the Invention
We have found that using DNA chip technology to immobilise all the necessary
DNA
sequences onto a solid phase format allows improved selection for zinc fingers
with
particular sequence specificity. Since at each stage of the selection
procedure, all possible
binding sites are present, specificity can be easily confirmed.
Accordingly, the present invention provides a library of DNA sequences
consisting of 4~'
2~ sequences, where N is greater than or equal to three, each sequence varying
from the other
sequences by comprising a different one of the 4'~ possible permutations of a
DNA
sequence of length N, wherein the library of DNA sequences is immobilised on a
solid
substrate.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-6-
The present invention also provides a method for designing a zinc finger
polypeptide
having specificity for a particular DNA sequence comprising a contiguous
sequence of N
nucleotides. where N is greater than or equal to three, which method
comprises:
(i) providing a zinc finger polypeptide, preferably by designing using a
rational design
method or by selection from a library;
(ii) producing the polypeptide;
(iii) determining the sequence specificity for the polypeptide by contacting a
library of
DNA sequences with the polypeptide and identifying the sequence or sequences
with
which the polypeptide binds to with greatest affinity;
(iv) if the sequence or sequences identified in step (iii) are not the desired
sequences,
making modifications to the amino acid sequence of the polypeptide, preferably
based on
rational design or by selection from a library, and repeating steps (ii) and
(iii),
wherein the libraw of DNA sequences consist of 4~ sequences, each sequence
varying from the other sequences by comprising a different one of the 4N
possible
permutations of the DNA sequence of length N, wherein the library of DNA
sequences is
immobilised on a solid substrate.
The present invention also provides a method for isolating a zinc finger
polypeptide having
specificity for a particular DNA sequence comprising a contiguous sequence of
N
nucleotides, where N is greater than or equal to three, which method
comprises:
(i) contacting a library of Garner organisms which express on their surface a
zinc finger polypeptide comprising variations in the amino acid sequence of
the zinc finger
DNA binding domain, with a library of DNA sequences; and
(ii) selecting those carrier organisms which express a zinc finger polypeptide
2~ that binds to the particular DNA sequence; and
(iii) optionally repeating selection steps (i) and (ii) with those carrier
organisms
selected in step (ii),
wherein the library of DNA sequences consist of 4N sequences, each sequence
varying from the other sequences by comprising a different one of the 4'~
possible
permutations of the DNA sequence of length N, wherein the library of DNA
sequences is
immobilised on a solid substrate.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
_7_
In another aspect the present invention provides a method for determining the
preferred
base recognition specificity of a zinc finger polypeptide, w -hich method
comprises
contacting a library of DNA sequences with the polypeptide, measuring the
affinity with
which the polypeptide binds to each of the sequences, and optionally ranking
the sequences
in order of the affinity with which the polypeptide binds,
wherein the library of DNA sequences consist of 4'~ sequences, each sequence
varying from the other sequences by comprising a different one of the 4~'
possible
permutations of the DNA sequence of length N, wherein the library of DN.~
sequences is
immobilised on a solid substrate.
In a preferred embodiment of the invention, each of the DNA sequences within
the library
occupies a discrete position on the solid substrate.
1 ~ The present invention also provides the use of a library of the invention
in a method for
designing a zinc finger polypeptide having specificity for a particular DNA
sequence.
The present invention further provides the use of a library of the invention
in a method for
isolating a zinc finger polypeptide having specificity for a particular DNA
sequence.
The present invention additionally provides the use of a library of the
invention in a
method for determining the preferred base recognition specificity of a zinc
finger
polypeptide.
2~ The DNA library may be arranged into two or more sub-libraries. Each sub-
library may
occupy a discrete position on the solid substrate. Preferably, each sub-
library comprises a
subset of the 4N sequences. In a preferred embodiment of the invention, the
library is
arranged in 4N sub-libraries, wherein for any one sub-library one base in the
DNA
sequence of length N is defined and the other N-1 bases are randomised.
According to a
further aspect of the invention, we provide such a sub-library.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
_g-
Brief Description of the Drawings
Fig 1. Overview of the protein engineering strategy.
Step 1. Two pre-made zinc finger phage-display libraries, Libl2 and Lib23,
contain
randomised DNA-binding amino acid positions in fingers 1 and 2 (black) or
fingers 2 and 3
(grey) respectively. Selections of 'one-and-a-half fingers from each master
library are
carried out in parallel using DNA sequences in which ~ nucleotides have been
fixed to a
sequence of interest.
Step 2. Zinc finger genes are amplified from the recovered phage using PCR and
sets of 'one-and-a-half fingers are paired to yield recombinant three-finger
DNA-binding
domains.
Step 3. The recombinant DNA-binding domains are cloned back into phage and
subjected to further rounds of selection, or immediately validated for binding
to a
composite 10 by DNA of pre-defined sequence.
Fig 2. Composition of the'bipartite' library.
(a) DNA recognition by the two zinc finger master libraries, Libl2 and Lib23.
The
libraries are based on the three-finger DNA-binding domain of Zif268 and the
putative
binding scheme is based on the crystal structure of the wild-type domain in
complex with
DNA (Pavletich, N. P. & Pabo, C. O. Zinc finger-DNA recognition: Crystal
structure of a
Zif268-DNA complex at 2.1 ~. Science 2~2, 809-817 (1991); Elrod-Erickson, M.,
Rould,
M. A., Nekludova, L. & Pabo, C. O. Zif268 protein-DNA complex refined at 1.6A:
a
model system for understanding zinc finger interactions. Structure 4, 1171-
1180 (1996)).
The DNA-binding positions of each zinc finger a.re numbered and randomised
residues in
the two libraries are circled. Broken arrows denote possible DNA contacts from
Lib 12 to
bases H'IJKLM and from Lib23 to bases MNOPQ. Solid arrows show DNA contacts
from
those regions of the two libraries that carry the wild-type Zif268 amino acid
sequence, as
observed in the crystal structure. The wild-type portion of each library
target site (white
boxes) determines the register of the zinc finger-DNA interactions, such that
the selected
portions of the two libraries can be recombined to recognise the composite
site
H'IJKLMNOPQ.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-9-
(b) Amino acid composition of the randomised DNA-binding positions on the a-
helix of each zinc finger. A subset of the 20 amino acids was included in each
DNA-
binding position. Note that positions 4 and 5 of F2 (LS) are specined by the
codons CTG
AGC, which contain the recognition site of the restriction enzyme DdeI
(underlined), used
as a breakpoint to recombine the products of the two libraries.
Table 1. Selection of DNA-binding domains to recognise the HIV-1 promoter.
(a) Nucleotide sequences from HIV-1 of the form 3'-HIJKLMNOPQ-5' as
recognised by phage clones A-G. Bases which are predicted to be bound by amino
acid
residues from Libl2 and Lib23, according to the model described in Fig. 2, are
shown in
bold black and grey, respectively. The position of base Q in each site is
numbered relative
to the transcription start site (+1) in the HIV promoter. Note that the
binding site for Clone
A contains ~ bases from the binding site of Zif268 (underlined); and that this
clone was
thus derived directly from Lib23, without the need for recombination.
1 ~ (b) Amino acid sequences of the helical regions from recombinant zinc
finger
DNA-binding domains that recognise HIV-1 sequences. The origin of the amino
acids is
" _ indicated by shading Libl2 and Lib23 residues in bold black and grey,
respectively. Clone
A, which was derived solely from Lib23, contains wild-type Zif?68 residues
(underlined).
(c) Apparent Kd for the interaction of the customised DNA-binding domains for
their cognate sequences as measured by phage ELISA.
Figure 3 shows a matrix specificity assay for seven zinc finger DNA-binding
domains
designed to bind sequences in the HIV-1 promoter. The seven constructs and
their
respective binding sites are labelled A-G. Binding of zinc fingers to 0.4 pmol
DNA per
501 well is plotted vertically from phage ELISA absorbance readings (A.~;o-
A6;o). Each
clone is tested using all seven DNA sequences but strong binding is only
observed to those
sequences against which they have been designed.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-10-
Detailed description of the invention
Although we have described the libraries and methods of our invention with
reference to the
selection, design, etc of a zinc finger polypeptide, it will be understood
that our invention
may be applied to other DNA or nucleic acid binding molecules, such as nucleic
acid binding
proteins or polypeptides (e.g., helix-turn-helix proteins), other nucleic
acids such as DNA,
RNA, or PNA (protein-nucleic acid), small molecules such as drug, an
intercalating
molecule, a major or minor groove binding molecule (such as distamycin), etc.
Thus, in a broad sense, our invention encompasses libraries and methods for
designing.
isolating, and determining the preferred base recognition specificity of any
nucleic acid
binding molecule.
A. DNA library
A DNA library of the invention is used to test the selectivity of a zinc
finger for a
nucleotide sequences of length N. Consequently, since there are four different
nucleotides
that occur naturally in genomic DNA, the total number of sequences required to
represent
all possible base permutations for a sequence of length N is 4N. However,
uracil, which
occurs in RNA, or other natural or non natural bases, may also be included,
either in
substitution for thymidine, or in addition. Thus, the DNA library of the
invention may have
SN sequences.
N is an integer having a value of at least three. That it to say that the
smallest library
2~ envisaged for testing binding to a nucleotide sequence where only one DNA
triplet is
varied, consists of 64 different sequences. However, N may be any integer
greater than or
equal to 3 such as 4, 5, 6, 7, 8 or 9. Typically, N only needs to be three
times the number
of zinc fingers being tested, optionally including a few additional residues
outside of the
binding site that may influence specificity. Thus, by way of example, to test
the specificity
of a protein comprising three zinc fingers, where all three fingers have been
engineered, it
may be desirable to use a library where N is at least 9. The DNA sequences in
the library
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
are typically immobilised at discrete positions on a solid substrate, such as
a DNA chip,
such that each different sequence is separated from other sequences on the
solid substrate.
The 4N possible permutations of the DNA sequence of length N sequence are
typically (but
need not be) arranged in sub-libraries. Preferably, the library is sub-divided
into 4N
sub-libraries. wherein for any one sub-library one base in the DNA sequence of
length N is
defined and the other N-1 bases are randomised. Thus in the case of a varied
DNA triplet,
there will be 12 sub-libraries.
The nucleotide sequence of length N may be generally, but need not be, part of
a longer
DNA molecule. Thus, the DNA sequences within the library may consist of
sequences
against which the binding of a binding molecule is tested (i.e., every base
position in the
DNA sequence is potentially involved binding to the binding molecule). An
example is a
library of 64 sequences of length 3 representing all possible targets for a
zinc finger motif.
1~
Alternatively, and preferably, the DNA sequences comprise other flanking
sequences
which are not directly relevant to or involved in binding. E~camples of such
sequences
include vector sequences, dimerisation sequences, or nucleic acid sequences
which are
capable of hybridising to other nucleic acid sequences to form double stranded
regions,
other binding targets, etc. The sequence and (where applicable, the binding
specificity) of
such flanking regions may be known or unknown.
For example, the DNA sequences may comprise one or more binding targets for
another
binding domain, whether this is a zinc finger domain or otherwise. Such
libraries are useful
2~ in designing, isolating, and determining the binding affinity and preferred
base recognition
specificity of a hybrid binder such as a zinc finger-homeodomain fusion
protein.
The nucleotide sequence of length N typically occupies the same position
within the longer
molecule in each of the varied sequences even though the sequence of N itself
may vary.
The other sequences within the DNA molecule are generally the same throughout
the
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-12-
library. Thus the library can be said to consist of a library of 4~' DNA
molecules of the
formula R~-[A/C/G/TJ.~N-R'', wherein R1 and R'' may be any nucleotide
sequence.
Preferably, each sequence is also represented as a dilution/concentration
series. Thus the
immobilised DNA library. may occupy Z4N discrete positions on the chip where Z
is the
number of different dilutions in the series and is an integer having a value
of at least 2.
The range of DNA concentrations for the dilution series is typically in the
order of 0.01 to
100 pmol cm'', preferably from 0.05 to 5 pmol crri 2. The concentrations
typically vary 10-
fold, i.e. a series may consist of 0.01, 0.1, 1, 10 and 100 pmol cm z, but may
vary, for
example, by 2- or 5-fold.
The advantage of including the DNA sequences in a dilution series is that it
is then possible
to estimate Kds for protein/DNA complexes using standard techniques such as
the
Kaleidagraph~ version 2.0 program (Abelback Software).
The DNA molecules in the library are at least partially double-stranded, in
particular at least
the nucleotide sequence of length N is double-stranded. Single stranded
regions may be
included, for example to assist in attaching the DNA library to the solid
substrate.
Techniques for producing immobilised libraries of DNA molecules have been
described in
the art. Generally, most prior art methods described how to synthesise single-
stranded
nucleic acid molecule libraries, using for example masking techniques to build
up various
permutations of sequences at the various discrete positions on the solid
substrate. U.S. Patent
No. 5,837,832, the contents of which are incorporated herein by reference,
describes an
improved method for producing DNA arrays immobilised to silicon substrates
based on very
large scale integration technology. In particular, U.S. Patent No. 5,837,832
describes a
strategy called "tiling" to synthesize specific sets of probes at spatially-
defined locations on a
substrate which may be used to produced the immobilised DNA libraries of the
present
invention. U.S. Patent No. 5,837,832 also provides references for earlier
techniques that may
also be used.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-13-
However, an important aspect of the present invention is that it relates to
DNA binding
proteins, zinc fingers, that bind double-stranded DNA. Thus single-stranded
nucleic acid
molecule libraries using the prior art techniques referred to above will then
need to be
converted to double-stranded DNA libraries by synthesising a complementary
strand. An
example of the conversion of single-stranded nucleic acid molecule libraries
to double-
stranded DNA libraries is given in Bulyk et al., 1999, Nature Biotechnology
17, 573-X77, the
contents of which are incorporated herein by reference. The technique
described in Bulyk et
al., 1999, ripically requires the inclusion of a constant sequence in every
member of the
library (i.e. within R~ or RZ in the generic formula given above) to which a
nucleotide primer
is bound to act as a primer for second strand synthesis using a DNA polymerase
and other
appropriate reagents. If required, deoxynucleotide triphosphates (dNTPs)
having a detectable
labeled may be include to allow the efficiency of second strand synthesis to
be monitored.
Also the detectable label may assist in detecting binding of zinc fingers when
the
immobilised DNA library is in use.
l~
Alternatively, double-stranded molecules may be synthesised off the solid
substrate and
each pre-formed sequence applied to a discrete position on the solid
substrate. An example
of such a method is to synthesis palindromic single-stranded nucleic acids -
see U.S. Patent
No. 5»6752. the contents of which are incorporated herein by reference.
Thus DNA may typically be synthesised in situ on the surface of the substrate.
However,
DNA may also be printed directly onto the substrate using for example robotic
devices
equipped with either pins or pizo electric devices.
2~ The library sequences are typically immobilised onto or in discrete regions
of a solid
substrate. The substrate may be porous to allow immobilisation within the
substrate or
substantially non-porous, in which case the library sequences are typically
immobilised on
the surface of the substrate. The solid substrate may be made of any material
to which
polypeptides can bind, either directly or indirectly. Examples of suitable
solid substrates
include flat glass, silicon wafers, mica, ceramics and organic polymers such
as plastics,
including polystyrene and polymethacrylate. It may also be possible to use
semi-permeable
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-1 ~l-
membranes such as nitrocellulose or nylon membranes, which are widely
available. The
semi-permeable membranes may be mounted on a more robust solid surface such as
glass.
The surfaces may optionally be coated with a layer of metal, such as gold,
platinum or
other transition metal. A particular example of a suitable solid substrate is
the
commercially available BiaCoreTM chip (Pharmacia Biosensors).
Preferably, the solid substrate is generally a material having a rigid or semi-
rigid surface. In
preferred embodiments, at least one surface of the substrate will be
substantially flat,
although in some embodiments it may be desirable to physically separate
synthesis regions
for different polymers with, for example, raised regions or etched trenches.
The solid
substrate may be a microtitre plate or bead. It is also preferred that the
solid substrate is
suitable for the high density application of DNA sequences in discrete areas
of typically
from 50 to 100 ~.m, giving a density of 10000 to 40000 cm-''.
1 ~ The solid substrate is conveniently divided up into sections. This may be
achieved by
techniques such as photoetching, or by the application of hydrophobic inks,
for example
teflon-based inks (Cel-line, USA). Where the solid substrate is a microtitre
plate, the
sections may conveniently comprise the wells of the microtitre plate. Each
well may
comprise a discrete DNA sequence of the library, or, in the case where the
library is sub
divided into sub-libraries, each well may comprise one or more sub-libraries.
Discrete positions, in which each different member of the library is located
may have any
convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
A discrete
position is commonly referred to as a ''spot". Each discrete position may
comprise,
2~ preferably consist of, one DNA sequence of the library. Thus, the discrete
position may
comprise a single molecule, or a number of DNA molecules of homogenous
composition.
The latter arrangement is advantageous in that the signal strength is likely
to be higher.
In an alternative embodiment, each discrete position comprises a number of DNA
molecules of heterogenous composition. In this embodiment, a number of
different DNA
sequences are immobilised at a discrete spot. Where the library is divided
into sub-
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-15-
libraries, as described above, preferably each discrete spot comprises the
sequences within
the sub-library. Thus, in a preferred embodiment, where the library is sub-
divided into 4N
sub-libraries, each of the sub-libraries is immobilised in a discrete position
on the solid
substrate. This embodiment is referred to as "multiplexing".
Attachment of the library sequences to the substrate may be by covalent or non-
covalent
means. The library sequences may be attached to the substrate via a layer of
molecules to
which the library sequences bind. For example, the library sequences may be
labelled with
biotin and the substrate coated with avidin and/or streptavidin. A convenient
feature of
using biotinylated library sequences is that the efficiency of coupling to the
solid substrate
can be determined easily. Since the library sequences may bind only poorly to
some solid
substrates, it is often necessary to provide a chemical interface between the
solid substrate
(such as in the case of glass) and the library sequences. Examples of suitable
chemical
interfaces include hexaethylene glycol. Another example is the use of
polylysine coated
1 ~ glass, the polylysine then being chemically modified using standard
procedures to
introduce an affinity ligand. Other methods for attaching molecules to the
surfaces of solid
substrate by the use of coupling agents are known in the art. see for example
W098/49»7.
Binding of zinc fingers to the immobilised DNA library may be determined by a
variety of
means such as changes in the optical characteristics of the bound DNA (i.e. by
the use of
ethidium bromide) or by the use of labelled zinc finger polypeptides, such as
epitope tagged
zinc finger polypeptides or zinc finger polypeptides labelled with
fluorophores such as green
fluorescent protein. Other detection techniques that do not require the use of
labels include
optical techniques such as optoacoustics, reflectometry, ellipsometry and
surface plasmon
resonance (SPR) - see W097/49989, incorporated herein by reference.
Binding of epitope tagged zinc finger polypeptides is typically assessed by
immunological
detection techniques where the primary or secondary antibody comprises a
detectable label.
A preferred detectable label is one that emits light, such as a fluorophore,
for example
phycoerythrin.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-16-
The complete DNA library is typically read at the same time by charged coupled
device
(CCD) camera or confocal imaging system. Alternatively, the DNA library may be
placed
for detection in a suitable apparatus that can move in an x-y direction, such
as a plate
reader. In this way, the change in characteristics for each discrete position
can be measured
automatically by computer controlled movement of the array to place each
discrete element
in turn in line with the detection means.
The detection means are capable of interrogating each position in the library
array optically
or electrically. Examples of suitable detection means include CCD cameras or
confocal
imaging systems.
Any of the immobilised DNA sequences of the library may be removed from the
solid
substrate for further manipulation. Thus, it may be desired to remove a
particular DNA
sequence which shows binding to a particular zinc finger, for example. Removal
from the
1 ~ solid substrate may be achieved by various means, for example, by elution
using an
appropriate solvent, by chemical or enzymatic cleavage, photochemical lysis
(e.g., by
application of laser energy), etc. The removed sequence may be amplified by
PCR, for
example.
B. Zinc fingers
A zinc finger binding motif is the a-helical structural motif found in zinc
finger binding
proteins, well known to those skilled in the art. The amino acid numbering
used throughout
is based on the first amino acid in the a-helix of the zinc finger binding
motif being position
2~ +1. It will be apparent to those skilled in the art that the amino acid
residue at position -1
does not, strictly speaking, form part of the a-helix of the zinc binding
finger motif.
Nevertheless, the residue at -1 is shown to be very important functionally and
is therefore
considered as part of the binding motif a-helix for the purposes of the
present invention.
The zinc finger polypeptide sequences to be tested and/or selected using the
methods of the
invention are typically obtained by modifying one or more amino acids residues
known to be
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-17-
important in binding specificity. Thus, for example, zinc finger pol5~peptide
sequences may
comprise a substitution at one or more of the followznQ positions: -l, +1, +2,
+3, +j +6 and
+8.
Zinger finger polypeptides may in one embodiment be tested individually using
the library
and methods of the invention. For example, it may be desired to determine the
preferred base
recognition specificity of a zinc finger polypeptide designed using rational
design techniques.
The term ''rational design" is intended to refer to the design of a zinc
finger sequence
according to one or more rules (recognition rules). Various rational design
techniques and
rules are known in the art, for example, as disclosed in W098/~30~7. Thus,
according to
W098/53~~7, a zinc finger may be designed to bind to a nucleic acid quadruplet
in a
target nucleic acid sequence, wherein binding to each base of the quadruplet
by an a-
helical zinc finger nucleic acid binding motif in the protein is determined as
follows: if
1 ~ base 4 in the quadruplet is G, then position +6 in the a-helix is Arg or
Lys; if base 4 in the
quadruplet is A, then position +6 in the a-helix is Glu, Asn or Val; if base 4
in the
quadruplet is T, then position +6 in the a-helix is Ser, Thr, Val or Lys; if
base 4 in the
quadruplet is C, then position +6 in the a-helix is Ser, Thr, Val, Ala, Glu or
Asn; if base 3
in the quadruplet is G, then position +3 in the a-helix is His: if base 3 in
the quadruplet is
A, then position +3 in the a-helix is Asn; if base 3 in the quadruplet is T,
then position +3
in the a-helix is Ala, Ser or Val; provided that if it is Ala, then one of the
residues at -1 or
+6 .is a small residue; if base 3 in the quadruplet is C, then position +3 in
the a-helix is Ser,
Asp, Glu, Leu, Thr or Val; if base 2 in the quadruplet is G, then position -1
in the a-helix is
Arg; if base 2 in the quadruplet is A, then position -1 in the a-helix is Gln;
if base 2 in the
2~ quadruplet is T, then position -1 in the a-helix is His or Thr; if base 2
in the quadruplet is
C, then position -1 in the a-helix is Asp or His: if base 1 in the quadruplet
is G, then
position +2 is Glu; if base 1 in the quadruplet is A, then position +2 Arg or
Gln; if base 1 in
the quadruplet is C, then position +2 is Asn, Gln, Arg. His or Lys; if base 1
in the
quadruplet is T, then position +2 is Ser or Thr.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-18-
These rules permit the design of a zinc finger binding protein specific for
any given nucleic
acid sequence. It has been found that position +2 in the helix is responsible
for determining
the binding to base 1 of the quadruplet. In doing so, it cooperates
synergistically with
position +6, which determines binding at base 4 in the quadruplet, bases l and
4 being
overlapping in adjacent quadruplets.
Although zinc finger polypeptides are considered to bind to overlapping
quadruplet
sequences, rational design rules such as the rules set out above allow
polypeptides to be
designed to bind to target sequences which are not multiples of overlapping
quadruplets.
For example, a zinc finger polypeptide may be designed to bind to a
palindromic target
sequence. Such sequences are commonly found as, for example, restriction
enzyme target
sequences. Furthermore, creation of zinc fingers which bind to fewer than
three nucleotides
may be achieved by specif~~ing, in the zinc finger, amino acids which are
unable to support
H-bonding with the nucleic acid in the relevant position. Advantageously, this
is achieved
1 ~ by substituting Gly at position -1 (to eliminate a contact with base 2)
and/or Ala at
positions +3 and/or +6 (to eliminate contacts at the 3rd or 4th base
respectively). The
contact with the final (3') base in the target sequence may be strengthened,
if necessary, by
substituting a residue at the relevant position which is capable of making a
direct contact
with the phosphate backbone of the nucleic acid.
In an alternative embodiment, a library of zinc finger polypeptides having
different amino
acids at one or more positions involved in binding specificity may be screened
(''empirical
selection") using the library and methods of the present invention and zinc
finger
polypeptides selected that bind to a target nucleotide sequence. Such a
library of sequences
may conveniently be obtained by random mutagenesis at particular positions to
produce a
phage display library using standard techniques (see W096/06166 for
construction of a
randomised Zif268 library).
Where a randomised zinc finger polvpeptide library is used, preferably the
zinc fingers are
randomised at one or more of, or may have a random allocation at some or all,
preferably all,
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-19-
of positions -l, +1, +2, +3, +5 +6, +8 and +9. More preferably, the zinc
fingers are
randomised at positions -1, +2, +3 and +6, and at least one of +1, +~ and +8.
The sequences may also be randomised at other positions (e.g. at position +9,
although it is
generally preferred to retain an arginine or a lysine residue at this
position). Further, whilst
allocation of amino acids at the designated "random" positions may be
genuinely random, it
is preferred to avoid a hydrophobic residue (Phe, Trp or TyT) or a cysteine
residue at such
positions.
Preferably the zinc finger binding motif is present within the context of
other amino acids
(which may be present in zinc finger proteins), so as to form a zinc finger
(which includes an
antiparallel p-sheet). Further, the zinc finger is preferably displayed as
part of a zinc finger
polypeptide, which polypeptide comprises a plurality of zinc fingers joined by
an intervening
linker peptide. Typically the library of sequences is such that the zinc
finger polypeptide will
1 ~ comprise two or more zinc fingers of defined amino acid sequence
(generally the wild type
sequence) and one zinc finger having a zinc finger binding motif randomised in
the manner
defined above. It is preferred that the randomised finger of the polypeptide
is positioned
between the two or more fingers having defined sequence. The defined fingers
will establish
the "phase" of binding of the polypeptide to DNA, which helps to increase the
binding
specificity of the randomised finger.
Preferably the sequences encode the randomised binding motif of the middle
finger of the
Zif268 polypeptide. Conveniently, the sequences also encode those amino acids
N-terminal
and C-terminal of the middle finger in wild type Zif268, which encode the
first and third zinc
fingers respectively. In a particular embodiment, the sequence encodes the
whole of the
Zif268 polypeptide. Those skilled in the art will appreciate that alterations
may also be made
to the sequence of the linker peptide and/or the (3-sheet of the zinc finger
polvpeptide.
Typically, the randomised sequence encoding zinc finger polypeptides are such
that the zinc
finger binding domain can be cloned as a fusion with the minor coat protein
(pIII) of
bacteriophage fd. Conveniently, the encoded polypeptide includes the
tripeptide sequence
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-20-
Met-Ala-Glu as the N terminal of the zinc finger domain, which is known to
allow expression
and display using the bacteriophage fd system. Desirably the polvpeptide
library comprises
106 or more different sequences (ideally, as many as is practicable).
C. Uses of the DNA library
Design and testing of custom zinc fingers
The immobilised DNA library of the present invention may conveniently be used
to verify the
results of rationally designing zinc fingers with desired specificity.
Typically a zinc finger
motif is designed as described above and then produced by recombinant or
synthetic means.
The zinc forger polypeptide is contacted with the immobilised DNA library and
binding
detected as described above. The specificity and affinity of the zinc finger
for the various
sequences in the library can then be determined. If the desired binding is not
seen then
1 ~ further modifications may be made to the zinc finger motif and the
screening process
repeated.
The use of automated peptide synthesisers and detection means together with
computer-
controlled equipment and software may allow the process to be fully automated
such that
when given a target sequence and rational design protocol, the process is
repeated
automatically until the desired result is obtained.
Screening for zinc finger polypeptides having specificity for one or more DNA
sequences.
2~ In another approach, a library of zinc forger polypeptides is contacted
with the DNA library
and the zinc fingers that bind to the target sequences) selected.
Conveniently, the zinc finger
library is in the form of a library of carrier organisms that express on their
surface a zinc
finger polypeptide. Typical earner organisms include phage and bacteria.
Alternatively, other means of phenotype-genotype linkage as known in the art
may be used.
For example, the libraries may be segregated into compartments or
microcapsules, as
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-2 ( -
described in W099/02671. This document discloses a method for isolating one or
more
Genetic elements encoding a gene product having a desired activity. Genetic
elements are
first compartmentalised into microcapsules, and then transcribed and/or
translated to
produce their respective gene products (RNA or protein) within the
microcapsules.
Alternatively, the genetic elements are contained within a host cell in which
transcription
and/or translation (expression) of the gene product takes place and the host
cells are first
compartmentalised into microcapsules. Genetic elements which produce gene
product
having desired activity are subsequently sorted. Polysome display techniques,
such as those
disclosed in WO00/27878, may also be applied to the libraries and methods of
our
invention.
More than one round of selection may take place, for example to confirm that
specificity of
zinc finger polypeptides selected in any particular round. Desirably at least
two, preferably
three or more, rounds of screening are performed.
1~
The library of zinc finger polypeptides need not necessarily be completely
random but may be
partially random, for example at certain positions only. The positions chosen
and the range
of different amino acids at any given position may be based on rational design
principles.
The two methods are not mutually exclusive and may both be used as part of a
design and
selection strategy. For example, it may be preferred to use the screening
method described
above as a precursor, to the rational design method described above. Thus in a
preferred
embodiment, that there is a two-step selection procedure: the first step
comprising screening
each of a plurality of zinc finger binding motifs (typically in the form of a
display library),
2~ mainly or wholly on the basis of affinity for the target sequence; the
second step comprising
comparing binding characteristics of those motifs selected by the initial
screening step, and
selecting those having preferable binding characteristics for a particular DNA
triplet.
The non-specific component of all protein-DNA interactions, which includes
contacts to the
sugar-phosphate backbone as well as ambiguous contacts to base-pairs, is a
considerable
driving force towards complex formation and can result in the selection of DNA-
binding
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
proteins with reasonable affinity but without specificity for a given DNA
sequence.
Therefore, in order to minimise these non-specific interactions when designing
a polypeptide,
selections should preferably be performed with low concentrations of specific
binding site in
a background of competitor DNA. and binding should desirably take place in
solution to
avoid local concentration effects and the avidity of multivalent phage for
ligands immobilised
on solid surfaces.
As a safeguard against spurious selections, the specificity of individual
phage should be
determined following the final round of selection.
Determining the preferred base recognition specificity of a zinc finger
polvpeptide
The immobilised DNA library of the present invention may be used in a general
sense to
determine the preferred base recognition specificity of a zinc finger
polypeptide, whether
1 ~ the zinc finger polypeptide be a naturally occurring zinc finger
polypeptide, or a fragment
thereof comprising a zinc finger motif, a zinc finger polypeptide identified
by a screening
procedure, such as the screening method of the invention, or a zinc finger
obtained by
rational design methods.
Typically, the zinc finger polypeptide of interest in contacted with the DNA
library as
described above and the extent of binding at each position on the immobilised
DNA library
determined. The results for each different sequence in the library may then be
placed in
order of the affinity with which the zinc finger polypeptide binds. The
resulting ranking
will provide a clear indication of the preferred base recognition specificity
of the zinc
finger polypeptide and may even be used to determine an optimal consensus
binding
sequence.
Uses of zinc finer motifs designed and/or selected by the methods of the
invention
Once suitable zinc finger binding motifs have been identified and obtained,
they will
advantageously be combined in a single zinc finger polypeptide. Typically this
will be
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-23-
accomplished by use of recombinant DNA technoloy; conveniently a phage display
system
may be used.
In a further aspect the invention provides a zinc finger polypeptide designed
and/or selected
by one or both of the methods defined above. Preferably the zinc finger
poly~peptide designed
by the method comprises a combination of a plurality of zinc fingers (adjacent
zinc fingers
being joined by an intervening linker peptide), each finger comprising a zinc
finger binding
motif. Desirably, each zinc finger binding motif in the zinc finger
polvpeptide has been
selected for preferable binding characteristics by the method defined above.
The intervening
linker peptide may be the same between each adjacent zinc finger or,
alternatively, the same
zinc finger polypeptide may contain a number of different linker peptides. The
intervening
linker peptide may be one that is present in naturally-occurring zinc finger
polypeptides or
may be an artificial sequence. In particular, the sequence of the intervening
linker peptide
may be varied, for example, to optimise binding of the zinc finger polypeptide
to the target
1~ sequence.
Where the zinc finger polypeptide comprises a plurality of zinc binding
motifs, it is preferred
that each motif binds to those DNA triplets which represent contiguous or
substantially
contiguous DNA in the sequence of interest. Where several candidate binding
motifs or
candidate combinations of motifs exist, these may be screened against the
actual target
sequence to determine the optimum composition of the polvpeptide. Competitor
DNA may
be included in the screening assay for comparison, as described above.
It is well within the capability of one of normal skill in the art to design a
zinc finger
polypeptide capable of binding to any desired target DNA sequence simply by
considering
the sequence of triplets present in the target DNA and combining in the
appropriate order zinc
fingers comprising zinc finger binding motifs having the necessary binding
characteristics to
bind thereto. The greater the length of known sequence of the target DNA, the
greater the
number of zinc finger binding motifs that can be included in the zinc finger
polypeptide. For
example, if the known sequence is only 9 bases long then three zinc finger
binding motifs can
be included in the polypeptide. If the known sequence is 27 bases long then,
in theory, up to
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-24-
nine binding motifs could be included in the polypeptide. The longer the
target DNA
sequence, the lower the probability of its occurrence in any given portion of
DNA.
Moreover, those motifs selected for inclusion in the polypeptide could be
artificially modified
(e.g. by directed mutagenesis) in order to optimise further their binding
characteristics.
Alternatively (or additionally) the length and amino acid sequence of the
linker peptide
joining adjacent zinc binding fingers could be varied, as outlined above. This
may have the
effect of altering the position of the zinc finger binding motif relative to
the DNA sequence of
interest, and thereby exert a further influence on binding characteristics.
Generally, it will be preferred to select those motifs having high affinity
and high specificity
for the target triplet.
Possible uses of suitably designed zinc finger polypeptides are:
1~ a) Therapy (e.g. targeting to double stranded DNA)
b) Diagnosis (e.g. detecting mutations in gene sequences: the present work has
shown
that "tailor made" zinc finger polypeptides can distinguish DNA sequences
differing by one
base pair).
c) DNA purification (the zinc finger polypeptide could be used to purify
restriction
fragments from solution, or to visualise DNA fragments on a gel - for example,
where the
polypeptide is linked to an appropriate fusion partner, or is detected by
probing with an
antibody).
In addition, zinc finger polypeptides could even be targeted to other nucleic
acids such as
single-stranded or double-stranded RNA (e.g. self complementary RNA such as is
present in
many RNA molecules) or to RNA-DNA hybrids, which would present another
possible
mechanism of affecting cellular events at the molecular level.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-25-
Examples
These examples show the use of the DNA libraries of the invention in designing
and/or
isolating a zinc finger polypeptide having a particular DNA sequence
specificity, as well as
in the determination of the preferred base recognition specificity of a zinc
finger
polypeptide.
General Materials and Methods for screening procedure using phaae library
1. Prepare DNA chips as in Bulyk et al., 1999, ibid.
2. Prepare a fresh phage culture for assay by innoculating 2m1 of 2xTY
containing
1 S pg/ml tetracycline with a single bacterial colony and incubating for 8 -
24 hours at 30°C.
1~ 3. Block chip surface for 1 hour at 20°C by adding 150 p1 PBS
containing 4% (w/v)
fat-free freeze-dried milk (Marvel).
4. Centrifuge phage cultures from step 2 on a benchtop microfuge for 10
minutes at
top speed to obtain clear phage-containing culture supernatant.
5. Prepare 200 p1 phage binding mixture for each assay by mixing 20 p1 phage
supernatant with 180 p.1 of PBS containing 2% (w/v) fat-free freeze-dried milk
(Marvel),
1% (v/v) Tween and.l p.g competitor nucleic acid, e.g. sonicated salmon sperm
DNA or
poly dIdC depending on the application.
6. Discard blocking mixture from chip and add phage binding mixture to chip.
Incubate for up to 1 hour at 20°C.
7. Remove unbound phage by washing chip 7 times with PBS containing 1% (v/v)
Tween followed by 3 washes with PBS.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-26-
8. Add PBS containing 2% (w/v) fat-free freeze-dried milk (Marvel) and 0.02%
(v/v)
biotin-conjugated anti-M13 IgG (Pharmacia Biotech). Incubate for 1 hour at
20°C.
9. Remove unbound antibody by washing chip 3 times with PBS containing 0.0~%
(v/v) Tween-20 followed by 3 washes with PBS.
10. Add a solution of streptavidin-phycoerythrin to the chip. Allow to bind
for 15
minutes at room temp. Remove unbound antibody by washing microtitre plate
wells 3
times with PBS containing 0.05% (v/v) Tween-20 followed by 3 washes with PBS.
11. Detection protocols as described in Bulyk et al., 1999, ibid
Example 1 - Use of a DN A chip to study a phage display library of the pZifZ68
middle
finger.
The DNA chip used in this protocol has 64 different features which correspond
to the 64
possible middle triplets of the Zif268 binding site. Each DNA binding site is
applied to the
chip at various densities, covering a roughly 100-fold range, from 0.04 to 4
pmol/cmz. The
DNA sequence synthesised on the chip is: 3'-cctggctaactgaactATATATGCG-NNN-
GCGATATAT-5'.
This sequence is attached to the chip at the 3' end of the strand, nucleotides
shown in
lowercase delineate the primer binding site used in Bulyk et al., 1999, ibid.
Screening of entire library on a chip
Every member of the Zif268 phage library (as described in Choo and Klug, 1994,
Proc Natl
Acad Sci U S A 91, 11163-11167) can be screened against every possible binding
site to
establish whether the phage display library has any limitations on DNA
recognition. This
helps ascertain the quality of a library. For instance we now know that the
Choo and Klug
library has certain sequence-restrictions which arise from the synergy of
fingers 2 and 3.
The overlap restricts binding to middle triplets with 5' G or T - this is
discussed fully in
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
Isalan et al., 1998, Biochemistry 37: 12026-33 and Isalan et al., 1997, Proc
Natl Acad Sci
U S A. 94: ~ 617-21.
Experiment: The library is applied onto a chip with the 6=I different
triplets. Binding is
observed only to those triplets with 5' G or T. Triplets with 5' A or C are
not bound: it is
concluded that the library is limited.
Following the selection process by screening on a chip.
Experiment 1: During the course of phage selections using the triplet TCC,
phage returned
from individual rounds of selection are applied on the chip. It is noted that
the signal for
binding to TCC increases in consecutive rounds of selection, but that there is
a higher
signal for binding to GAC. It is concluded that (since the phage library is
not capable of
binding to triplets with 5' T) selection using the oligo with middle triplet
TCC (~'-tatata
GCG-TCC-GCG-tatata-3'; putative binding site underlined) has selected fingers
that bind
1~ quite tightly to a frame-shifted sequence on the complementary strand (3'-
atatat-CGC-
AGG-CGC-atatat-5'; putative binding site underlined). Note that the frameshift
means that
finger 1 is forced to recognise the triplet GCT rather than GCG, which is
suboptimal.
When the triplet GAC is offered to these fingers in the context of the correct
binding site
for fingers 1 and 3, binding is optimal and a higher signal is obtained. When
the amino acid
sequences of zinc fingers isolated from separate selections using the triplets
TCC and GAC
are compared it is seen that the same fingers have been isolated, thus
confirming the above
hypothesis.
Experiment 2: While carrying out phage selections using the triplet GCG, phage
returned
2~ from individual rounds of selection are applied on the chip. At each round
the signal for
GCG is seen to increase relative to the other triplets, demonstrating
enrichment. By round 3
it is seen that there is appreciable binding to GCG and very little binding to
all other
triplets, except for binding to GTG which is also seen. It is concluded that 3
rounds of
selection are sufficient to eliminate binders of the other 62 triplets. It is
also concluded that
the selection has either (i) produced fingers which cannot discriminate
between GCG and
GTG, or (ii) produced a mixed population of fingers some of which bind GCG and
others
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-28-
GTG. To solve these problems the selection is repeated, including a specific
competitor to
eliminate GTG binders.
Studying sequence specificity and affinity of (individz~al) clones on a chip.
Experiment 1: After the GCG selection is repeated, including a specific
competitor to
eliminate GTG binders, two different ZnF clones [a-helix seq (A) RGPDLARHGR
and
(B) REDVLIRHGK] are isolated and sequenced. These are analysed separately on
the chip.
Clone A is seen to bind specifically to the feature containing GCG - it is
concluded that
this clone is highly sequence-specific. Clone B lights up features with both
GCG and GTG
- this clone is bispecific. From the relative intensities of binding to the
gradients of DNA
on the chip it is concluded that the two clones have roughly equal affinity
for the GCG site.
and it is deduced that this affinity is in the nanomolar range.
Studying spacing requirements for zinc f nger binding
1 ~ DNA arrays are synthesised of the form 3' cctggctaactgaactATATAT-GCG-GGT-
GCG-
Nx-GCG-CAG-GCG-ATATAT S', i.e. that contain variable nucleotide spacing (of 0
to
bp) between two 3-finger binding sites. Features are also included that
contain one or
the other binding site, but not both in a head to tail orientation as above. A
6-finger protein
is constructed comprising a fusion between wild-type Zifz68 three-fingers and
a three-
20 finger protein selected from the Choo and Klug library to bind GAC, linked
by the linker H
(zinc chelating)-LRQKDGERP-Y (hydrophobic core) where H and Y are the last and
first
structural elements of two adjacent fingers. The protein is applied to the
chip and
appreciable binding is seen to those features in which the spacing (Nx) is
from 0 to 3
nucleotides, but no binding is observed to features where the spacing is
greater than 7. It is
2~ concluded that the linker design restricts binding to short spacings
between adjacent
binding sites. From the relative intensities of binding to the gradients of
DNA on the chip it
is concluded that the protein binds to those features which contain both
binding sites
spaced by 0 to 3 by much more tightly (100-fold tighter) than to features
containing only
one binding site - it is concluded that the protein shows high discrimination
for the
composite site relative to either half site.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-29-
Example 2: Construction of DNA-binding domains by phage display
A bipartite-complementary' system for the construction of DNA-binding domains
by phage
display may be used (Fig. 1). This system comprises rivo master libraries,
Libl2 and Lib23,
each of which encodes variants of a three-finger DNA-binding domain based on
that of the
transcription factor Zif268 (Pavletich, N. P. & Pabo, C. 0. Zinc finger-DNA
recognition:
Crystal structure of a Zif268-DNA complex at 2.1 ~. Science 2~2, 809-817
(1991);
Christy, B. A., Lau, L. F. & Nathans, D. A gene activated in mouse 3T3 cells
by serum
growth factors encodes a protein with "zinc finger" sequences. Proc. Natl.
Acacz' Sci. USA
8~, 7857-7861 (1988).). The two libraries are complementary because Libl2
contains
randomisations in all the base-contacting positions of F l and certain base-
contacting
positions of F2, while Lib23 contains randomisations in the remaining base-
contacting
positions of F2 and all the base-contacting positions of F3 (Fig. 2a). The non-
randomised
DNA-contacting residues carry the nucleotide specificity of the parental
Zif268 DNA-
binding domain.
The design of the bipartite system features at least two modifications to the
conventional
zinc finger engineering strategies. As described above, each library contains
members that
are randomised in the a-helical DNA-contacting residues from more than one
zinc finger.
We have shown that the simultaneous randomisation of positions from adjacent
fingers
results in selected zinc finger pairs that can achieve comprehensive DNA
recognition, i.e.
bind DNA without significant sequence limitations.
The proteins produced by these libraries are therefore not limited to binding
DNA
sequences of the form GNNGNN..., as is the case with many prior art libraries
(eg. 9, 13,
20).
The repertoire of randomisations does not encode all 20 amino acids, rather
representing
only those residues that most frequently function in sequence-specific DNA
binding from
the respective a-helical positions (Fig 2b). Excluding the residues that do
not frequently
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-30-
function in DNA recognition advantageously helps to reduce the library size
and/or the
'noise' associated with non-specific binding members of the library.
Phage libraries for use in the present invention are prepared as follows.
Genes for the two zinc finger phage display libraries (Libl2 and Lib23) are
assembled from
synthetic DNA oligonucleotides by directional end-to-end ligation using short
complementary DNA linkers. In order to include only the amino acids shown in
Fig. 2b, a
large number of appropriately randomised oligonucleotides (each encoding a
subset of a
few amino acids) are used in combinations to assemble the gene cassettes.
These are
amplified by PCR, digested with SfiI and NotI endonucleases, and ligated into
the phage
vector Fd-Tet-SN (Pavletich, N. P. & Pabo, C. O. Zinc finger-DNA recognition:
Crystal
structure of a Zif268-DNA complex at 2.1 ~. Science 2~2, 809-817 (1991)),
E. coli TG1 cells are transformed with the recombinant vector by
electroporation and
plated onto TYE medium (1.5 % (w/v) agar, 1 % (w/v) Bactotryptone, 0.~ % (w/v)
Bactoyeast extract, 0.8 % (w/v) NaCI) containing 1 ~ ~g/ml tetracycline.
The theoretical library sizes of Libl2 and Lib23 are approx. 4.9 x 106 and
approx. 2.1 x
106, respectively (Fig. 2b).
Approximately twice these numbers of bacterial transformants are obtained for
the
respective libraries.
Example 3: Production of DNA-binding domains that target the HIV-1 promoter
Phage selections from the two master libraries described in Example 2 (Libl2
and Lib23)
are performed using the generic DNA sequence 3'-HIJKLMGGCG-5' for Libl2, and
3'-
GGCGMNOPQ-5' for Lib23, where the underlined bases are bound by the wild-type
portion of the DNA-binding domain and each of the other letters represents any
given
nucleotide (Fig. 2a).
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-31-
A number of sites in the well-characterised promoter of HIV-1 are targeted.
In this example, the two zinc finger libraries (Libl2 and Lib23) are subjected
to selection in
parallel, the nucleotide sequences used (ie. HIJKL/I~1NOPQ) being from HIV-1
between
positions -80 and +60 (see Table 1/Fig. 3).
Tetracycline resistant bacterial colonies are transferred to 2 ~c TY liquid
medium (16 g/litre
Bactotryptone, 10 g/litre Bactoyeast e~ctract, 5 g/litre NaCI) containing 50
~M ZnCh and
1 ~ ~Jml tetracycline, and cultured overnight at 30°C in a shaking
incubator.
Cleared culture supernatant containing phage particles is obtained by
centrifuging at 300 a
for 5 minutes.
1 ~ One picomole of biotinylated DNA target site is bound to streptavidin-
coated tubes
(Roche), in ~0 p,1 PBS containing 50 p.M ZnCh. Bacterial culture supernatant
containing
phaQe is diluted 1:10 in selection buffer (PBS containing 60 ~M ZnCh 2 % (w/v)
fat-free
dried milk (Marvel), 1 % (v/v) Tween, 20 mg/ml sonicated salmon sperm DNA),
and 1 ml
is applied to each tube. Binding reactions are incubated for 1 hour at
20°C, after which the
tubes are emptied and washed 20 times with PBS containing 50 p.M ZnCh, 2 %
(w/v) fat-
free dried milk (Marvel) and 1 % (v/v) Tween.
Retained phage are eluted in 0.1 M triethylamine and neutralised with an equal
volume of 1
M Tris-HCI (pH 7.4). Logarithmic-phase E. coli TG1 are infected with eluted
phage, and
2~ cultured overnight at 30°C in 2 0o TY medium containing 50 p.M ZnCh
and 1 ~ ~g/ml
tetracycline, to amplify phage for further rounds of selection.
After ~ rounds of selection, E. coli TG1 infected with selected phage are
plated and
individual colonies are picked and cultured in liquid medium to prepare phage
for ELISA
DNA-binding assays (Choo, Y. & Klug, A. Selection of DNA binding sites for
zinc fingers
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-32-
using rationally randomised DNA reveals coded interactions. Proc. lVatl. Acad.
Sci. U.S.A.
91, 11168-11172 (1994); Example 4).
Clones which recognise their target site may be retained for subsequent
recombination of
the two complementary halves recovered from Libl2 and Lib23 to produce
molecules
having high affinity for the HIV-1 promoter.
Eight DNA-binding domains are produced (Table l, clones A-G; Clone H (HIV A')
binds
5'-GCC TGG G(T/C)G-3' having the sequences Fl-RSDVLTR; F2-RSDHLTT; F3-
DYSVRKR).
Six (clones B-G) are engineered according to the full 'bipartite' protocol,
while one protein
(clone A) is derived directly by selection from Lib23. This illustrates a
further use of the
master libraries, namely to select zinc finger domains that bind DNA sequences
containing
1 ~ the motif 5'-GCGG-3' or 5'-GGCG-3'.
Four proteins have binding sites that are dispersed upstream of the
transcription initiation
site (clones A-D), including two that flank the TATA box (clones C-D). Another
three
proteins bind to a cluster of sites at the beginning of the ORF, within the
coding region for
TAR (clones E-G). Clone H (HIV A') binds between the sites for HIV A and HIV
B.
As the randomisations in the master libraries are restricted to amino acids
with validated
roles in DNA recognition, many of the recombinant DNA-binding domains make use
of
contacts that are consistent with the zinc finger-DNA 'recognition code'
(Choo, Y. & Klug,
A. Physical basis of a protein-DNA recognition code. Curr. Opin. Str. Biol. 7,
117-12~
(1997).): e.g. the well-known RXD motif found at the N-terminus of many zinc
finger a-
helices is selected in clones A, B and G.
In summary, using our selection method we produced seven DNA-binding domains
binding different loci in the genome of HIV-1 between positions -80 and +60
(Table 1).
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-33-
Example 4: ELISA DNA Binding Assays
As noted above, the immobilised DNA library of the present invention may be
used to
verify the binding ability of rationally designed zinc fingers, or they may be
used to screen
for zinc fingers having .specificity for one or more DNA sequences, or to
determine the
preferred base recognition specificity of a zinc finger. The binding
specificity of the zinc
finger sequences to a particular sequence or sequences within the immobilised
library may
be determined by any suitable binding assay as known in the art, for example,
an ELISA
assay as follows:
Equipment and reagents
~ Sterile, round-bottom, 200 ~l, 96-well plates for tissue culture (Costar,
Corning USA)
~ 2 x TY (Bacto tryptone, 16.0 g/1; Bacto yeast extract, 10.0 g/1; NaCI, 5.0
g/1)
~ Tetracycline
~ Zinc chloride, 1 M
~ Streptavidin-coated microtitre well plates (Roche).
~ PBS (10 x stock solution: NaCI, 80 g/1;KC1, 2Q/1; Na~HP0.~.7H~0 11.5
g/I;KH~PO.~, 2
g/1)
~ Fat-free freeze-dried milk (Marvel; Premier Brands UK Ltd.)
~ Tween-20
~ Sonicated salmon sperm DNA (lOmJml)
~ Horseradish peroXidase-conjugated anti-M13 IgG (Pharmacia Biotech)
~ ELISA developer solution [0.1 M Na (CH;.COO), pH 5.5; 3', 3', 5' S'-
tetramethylbenzidine (TMB; Sigma), 0.5 mg/ml; dimethyl sulphoxide (DMSO), 1%
(v/v); H202, 0.05% (v/v)J
~ Sulphuric acid, 1 M
~ ELISA plate reader
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-34-
Method
1. Pick single bacterial colonies containing phage clones derived from library
selections. Use a sterile toothpick to transfer colonies to wells in sterile
round-
bottom plates containing 150 ~l of 2 x TY ~g/ml tetracycline. As a positive
control,
use one well to grow phage displaying the wild-type DNA-binding domain.
Certain nucleic acid-binding domains may require supplements to the growth
medium. Zinc fingers, for example, are stabilised by 50 ~I~IZnCl2 in all media
and
ELISA binding and wash buffers. Incubate plates with orbital mixing at 250
rpm,
for 16 hours at 30°C.
2. Add biotinylated nucleic acid target sites (typically between 0 - 5 pmol)
in 50 p1
PBS to streptavidin-coated microtitre wells (Roche). For the positive control,
add
an appropriate amount of the wild-type binding site to one well. Use a
negative
control well, containing PBS only, to measure the ELISA background. Bind DNA
sites for 1$ minutes at 20°C.
3. To each well, add 1~0 ~l of PBS containing 4% (w/v) Marvel as a blocking
agent.
Leave blocking reaction for 1 hour at 20°C.
4. Prepare phage supernatant by centrifuging the 96-well culture plates at
3700 g for
15 minutes, in an appropriate swinging-bucket centrifuge.
5. Dilute phage supernatant 1:10 in 1 ml PBS containing 2% (w/v) Marvel, 1%
(v/v)
Tween-20 and 20 pg/ml sonicated salmon sperm DNA.
6. Discard blocking solution from the nucleic acid-coated wells and apply 50
p.1 of the
diluted phage supernatant solution. Incubate for 1 hour at 20°C.
7. Discard the binding mixture and wash the well 7 times with 200 p.1 PBS,
containing
1% (v/v) Tween-20. Wash a further 3 times with 200 ~l PBS alone.
8. To each well, add 50 p.1 of PBS containing 2% (v/v) Marvel and a 1:5000
dilution
of horseradish peroxidase (HRP)- conjugated anti-M13 IgG antibody (Pharmacia
Biotech). Incubate at 20°C for 1 hour.
CA 02382541 2002-03-28
WO 01/25417 PCT/GB00/03765
-35-
9. Discard the antibody binding mixture and wash the wells 3 times with 200 ~l
PBS,
containing 0.05% (v/v) Tween-20. Wash a further 3 times with 200 ~l PBS alone.
10. Develop the ELISA using 100 p.1 of HRP substrate such as the TMB-based
ELISA
developer solution described above. Stop the colorimetric reaction after
approximately 5 minutes; for TMB add 100 ~l of 1 H H~SO:~. Quantitate the
ELISA
signals immediately using a spectrophotometer fitted a microtitre plate
reader.
Although the protocol recited above relates to phage clones expressing zinc
fingers which
have been selected, the protocol may readily be adapted to assay interactions
between
specific zinc finger polypeptides and DNA substrates.
The ELISA DNA binding assay described above may be used to determine the
binding
specificity of a particular zinc finger, or a series of zinc fingers.
Similarly, either a single
DNA sequence or a series of DNA sequences may be tested.
Figure 1 shows the results of such an ELISA assay. Seven zinc finger DNA-
binding
domains are designed to bind sequences in the HIV-1 promoter. The seven
constructs and
their respective binding sites are labelled A-G, and each clone is tested
using all seven
DNA sequences. Binding of zinc fingers to 0.4 pmol DNA per 501 well is plotted
vertically from phage ELISA absorbance readings (A:~;o-A6;o). As can be seen
from the
figure, strong and specific binding of each zinc finger is only observed to
the DNA
sequence against which it has been designed. See also Table 1.
All publications mentioned in the above specification are herein incorporated
by reference.
Various modifications and variations of the described methods and system of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly
limited to such specific embodiments. Indeed, various modifications of the
described
modes for carrying out the invention which are obvious to those skilled in
molecular
biology or related fields are intended to be within the scope of the following
claims.
