Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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WO 96/27680 PCT/EP96/00893
Sequence-Specific Detection of Nucleic Acids
Subject matter of the invention is a solid carrier having two or more nucleic acid analogs
with di~erenl base sequences bound to predetermined sites on its surface. The invention
also addresses a method for the detection of nucleic acids using a carrier of this nature.
Sample analysis has undergone rapid development in recent dec~des While analytes were
initially detected primarily by means of their reaction with conventional chemical re~gent~,
and later on with enzymes, tests that utilize the immlln~logical characteri~tics of the analyte
have become the standard recently, especially in m~lic~ p;nostics. This is especially true
in the field of infectious diseases. However, immllnf~logical procedures can basically only
detect analy-tes with which imm-lnologically active compounds such as antigens or
antibodies play a role. These procedures have resulted in promising poten~ial applications
for many infections caused by viruses or bacteria. Genetic ~ e~eS or predispositions that
are not expressed as a change in protein patterns--or only to an in~llffici~nt extent--are
either diffi'-,lllt or impossible to detect using immllnological procedures, however. Nucleic
acids have therefore ~ecell~ly become the object of detectinn in many cases. The presence of
certain nucleic acids can infer the presence of an infectious agent or the genetic condition of
an organism. Detection procedures based on the presence of special nucleotide sequences in
particular were f~ilit~ted recently when methods for the amplification of nucleic acids that
are present in small numbers became available. Due to the large quantity of sequence
information and the fact that two nucleotide sequences with completely di~erellL functions
often differ by just one base unit, the specific detection of nucleotide sequences still poses a
considerable ch~ nP~e for reagents and analytical methods that are based on the detection
of nucleic acid sequences. In addition, the nucleotide sequences are often not even known,
but rather are det~rmined for the first time in the nucleic acid detectinn method itself.
A method for the detection of nucleotide sequences of the E~A gene is described in
EP-B-0 237 362 with which a clinically relevant point mutation can be detected In this
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method, an oligonucleotide that is bound to a membrane and has a nucleotide sequence that
is exactly complçm~nt~ry to one of the two nucleic acids to be di~er~ ted is brought in
contact with the sample. While certain conditions are ..~ e~l~ only that nucleic acid that
is exactly complementary binds to the oligoml~.lçoti(le that is bound to the solid phase, and
can be detected.
A method is described in Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1989) in which a large
number of oligonucleotides that are bound to di~relenl, predeterminecl sites of a nylon
membrane by means of poly-dT are used for the simlllt~neous detection of all known allelic
variants of an amplified region of a nucleic acid.
A method is described in US-A 5,202,231 in which the sequence of a nucleic acid can be
det~rrnined theoretically by bringing oligonucleotides having a predetermined, known
sequence in contact with a sample of the unknown nucleic acids under hybridization
conditions. This requires that all possible permutations of the nucleotide sequence be
immobilized on known sites of a solid phase. By determining the sites to which the nucleic
acids cont~ining the sequence to be deterrnined hybridize, it can theoretically be determined
which sequences are present in the nucleic acid.
Prior art in the field of the analysis of genetic polymorphisms using "oligonucleotide arrays"
is described in Nucleic Acids Research 22, 5456-5465 (1994) and Clin. Chem. 41/5, 700-6
(1995).
The main problem with the prior art is the fact that the melting temperatures of the selected
sequence-specific oligonucleotides cnnt~inin~ the nucleic acids to be sequenced or detected
are di~ele"~. To remedy this sit-l~tiQn~ one has to perform the complex method of selecting
the length ofthe oligonucleotide and its base composition, and opl;...;,;i-g the position of
the mi.cm~trhes within the oligonucleotide as well as the salt concentration of the
hykri~i7~tion complex. In many cases, however, it is practically impossible to
.~imlllt~n~.ously tli.~tin~ h closely related sequences from each other. The hybridization
temperature is another critical parameter. Variations of as little as 1 to 2 ~C can change the
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intensity or produce false-negative results. Incorrect analytical results based on the presence
of point mutations have serious impliç~tiQn~ for diagnosis.
The object of this invention was, thereIore, to provide an ~ltern~tive method for the
sequence-specific detection of nucleic acids and to provide suitable m~teri~l~ for this
method.
This object was accomplished by providing a solid carrier having two or more nucleic acid
analogs with di~ele-l~ base sequences bound to predetermined sites on its surface. Another
object of the invention is a method for the sequence-specific detection of a nucleic acid
using this solid carrier.
A"solid carrier" as described by this invention refers to an object that has a surface that is
so broad that specific areas can be di~tin~lish~d upon it. This surface is preferably flat and
larger than 5 mmZ, and is preferably between 10 mm2 and approx. 100 cm2. The carrier
material is not liquid or gaseous, and preferably dissolves either not at all or incompletely in
the sample fluids or reaction preparations that are used to immobilize nucleic acids to the
surface. Examples of such materials are glass, plastics (e.g. poly~Lyl~i, e, polyamide,
polyethylene, polypropylene), gold, etc. The m~teri~l does not n~cess~rily have to be
completely solid itself, but rather can be made solid by the ~tt~hmPnt of supporting
m~t~ri~
The external shape of the solid carrier basically depends on the method used to detect the
presence of nucleic acids on this solid carrier. It has proven to be applopliate, for in~t~nc.
to select a basically planar form, e.g. a chip.
Solid carriers that are especially suitable are, therefore, polystyrene chips that are from 1 to
5 mm thick and have a surface area of from 1 to 5 cm2, for in~t~nce Polyamide membranes
that are 4 x 2.5 cm2 in size have proven to be especially well-suited for use with this
invention. Two or more nucleic acid analogs having different base sequences are bound to
different sites of the surface of this carrier. These sites or regions preferably do not overlap
with each other. They are preferably separated from each other by regions on the surface to
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which no nucleic acid analogs are bound. The sites to which the nucleic acid analogs are
bound are referred to as "binding regions" below. The binding regions can have di~renl
shapes. These shapes are basically det~rmin~d by the method of m~mlf~ctllring the solid
carrier or by the method used to det~ormine the nucleic acid analogs in the binding regions.
The minimllm size of the binding regions is basically detprmined by the instrument with
which the event--the binding of a nucleic acid to nucleic acid analogs of a region--is
detecte~l Instruments are already available that can detect binding to regions that are
approx. 1 mm in size. The upper limit of the size of the binding regions is determined by
cost effectiveness and h~ntlling considerations.
The size of the binding regions is also basically determined by the methods used to apply the
nucleic acid analogs to the surface. Such methods will be described later.
The number of binding regions on the solid carrier depends on the intended use of the solid
carrier. In the simplest case, just two binding regions are needed to detect a certain point
mutation. In this case, a binding region contains nucleic acid analogs that have a base in the
position at which the point mutation is to be detected This base is compl~m~ont~ry to the
base in the position of the normal sequence. The other binding region, on the other hand,
contains a nucleic acid analog that has a base in the corresponding position that is
complement~ry to the base ofthe ml-t:~ted sequence. In another case, two nucleic acids or
nucleic acid sequences that are only slightly related to each other can be detected
~iml~lt~neously using a solid carrier that has two completely di~e~ nucleic acid analogs
bound to its surface.
"Nucleic acid analogs" refer to non-naturally occurring molecules that can detect nucleic
acids by means of base pairings. They therefore contain a specific base sequence that is
completely complem~nt~ry to the base sequence of a nucleic acid to be detecte~1 The base
sequence is therefore preferably composed of two naturally occllrring nucleobases. As long
as the specificity of the base pairing is not lost, modifications to the nucleobases are also
allowed, however.
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Those nucleic acid analogs are considered compl~ment~ry to a nucleic acid that have a base
sequence that forms hydrogen bridges with a base sequence of the nucleic acid per the
principle of base pairing when it is bound to the nucleic acid. This sequence is preferably at
least 8 bases long and, more preferably, between 8 and 25 bases long.
The nucleic acid analogs are further defined by the fact that they are structurally di~ert;l,~
from nucleic acids, at least in terrns of the backbone. The "backbone" in nucleic acids or a
nucleic acid analog refers to a structure that is basically composed of i~l~ntic~l units that
each contain a base. In naturally occ~ ng nucleic acids, the backbone is a sugar phosphate
backbone. This backbone is structurally modified in nucleic acid analogs, e.g. in that the
sugar or phosphate portion is completely or partially replaced with other çhPmiczil units
such as non-cyclic components. Basically, icl~ntic~l units can also replace each other in the
backbone.
A few characteristics of nucleic acid analogs are described below to f~rilit~te the selection
of nucleic acid analogs that are suitable for use with this invention. It is advantageous for
nucleic acid analogs to have a higher affinity to sequence-complementary nucleic acids than
an oligonucleotide with an idçn~ic~l base sequence. In addition, those nucleic acid analogs
are plere-led that carry fewer charges than a corresponding oligonucleotide ofthe same
length, or that can compensate charges with the opposite charges. Basically, uncharged
nucleic acid analogs are especially prefelled. Especially p-efelled nucleic acid analogs are
those whose affinity to complçmPnt~ry nucleic acids basically does not depend on the salt
content of the hybridization complex.
The nucleic acid analogs that are especially suitable are the nucleic acid analogs described in
WO 92/20702 and WO 92/20703 ~Peptide Nucleic Acid, PNA, e.g. Nature 365, 566-568(1993) and Nucl. Acids Res. 21, 533~-5 (1993)). These patent applications are referred to
for the description of the structure of the nucleic acid analogs. Preferred nucleic acid
analogs are compounds that have a polyamide backbone that contains a number of bases
bound along the backbone, with each base bound to a nitrogen atom of the backbone.
Nucleic acid analogs should also include compounds, however, like those described in EP-
A-0 672 677. Additional nucleic acid analogs are described in Recueil 91, 1069-1080
~3~ no~
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(1971), Methods in Molecular Biology 29, 355-389 (1993), Tetrahedron 31, 73-75 (1975),
J. Org. Chem. 52, 4202-4206 (1987), Nucl. Acids Res. 17, 6129-6141 (1989), Unusual
Properties of New Polymers (Springer Verlag 1983), 1-16, Specialty Polymers (Springer
Verlag 1981), 1-51, WO 92/20823, WO 94/06815, WO 86/05518 and WO 86/05519.
Additional nucleic acid analogs are described in Proc. Natl. Acad. Sci. USA 91, 7864-7868
(1994), Proc. Nat. Acad. Sci. USA 92, 6097-6101 (1995) and J. Am. Chem. Soc. 117,
6140-6141 (1995). The nucleic acid analogs described are from 8 to 30 bases long, while a
length offrom 10 to 25 bases is especially advantageous. The nucleic acid analogs named
are bound directly or indirectly to the surface of the solid carrier. The type of binding
basically depends on which reactive groups are available for binding on the solid carrier, and
which reactive groups are available for binding to the nucleic acid analog without restricting
the ability of the nucleic acid analogs to bind to a complçm~nt~ry nucleic acid. The type of
binding also depends on whether the intent is to ~iml~lt~neously bind the nucleic acid
analogs to different sites, or to build upon them. It can also be applupliate to cover the
surface of the solid carrier with a layer of a material that has a greater ability to bind, or to
activate the surface by means of a çhemic~l reaction. Reactive groups on the surface of a
solid carrier are usually selected from the group -OH, -NH2 and SH. Reactive groups of
nucleic acid analogs are preferably selected from the group -~X N H2,-S X -COO X -
SO3H and -PO3H2.
In an especially preferred embodiment, the reactive groups of the surface and the nucleic
acid analog are covalently bound to each other, especially by means of a linker that is more
than 15 atoms and less than 200 atoms long. A "linker" refers to a portion of a molecule
that basically has the function of removing the nucleic acid analogs that are sterically
available on the surface of the solid carrier. A linker is usually selected that has hydrogen
atoms (e.g. in alkylene units) and numerous heteroatoms (e.g. -O- or -NH- or -NR-) that
f~.ilit~te solvation. The linker preferably contains one or more ethylene oxy units and/or
peptide groups. In an especially plerell~d embodiment, the linker contains one or more units
as described in DE-A 3924705. Especially plerel-ed are the units described as an example
which is referred to as Ado (8-amino-3,6-dioxa-octanic acid), below. A slight dependence
of the binding of nucleic acids to the PNA surface can be reduced by using longer linkers
between PNA and the solid carrier.
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The nucleic acid analog that is bound to a site can also be a mixture of two or more analogs
having difrelen~ but known sequences. This can reduce the number of sites required for a
multiple dete, ,,,i~ ion
The surface of the carrier is preferably not charged, and is preferably hydrophilic. The
invention demonstrated that the use of basically uncharged s~ ce~e is an advantage when
cletecting nucleic acids.
A solid carrier loaded with nucleic acid analogs at di~elenL sites as provided by this
invention can be m~mlf~ctllred in different ways. In one embodiment, suitable qu~ntities of
solutions that each contain dirre~ nucleic acid analogs are applied to di~le-ll sites on the
surface of the solid carrier, e.g. via pipette. The liquid sarnples should not mix with each
other on the surface of the solid carrier. This can be accomplished, for in.~t~nr.e, by locating
the application sites far apart from each other or by using a hydrophobic barrier to stop the
exr~n.eiQn of the liquid between the various sites. Either the nucleic acid analogs or the
surface of the solid carrier is preferably activated for the reaction. This activation can be
achieved, for in.et~nre, in that one of the groups described above is activated by the creation
of a reactive species. In the case of a carboxyl group, this would be an activated ester (e.g. a
N-hydlo~y srlcrinimide ester) that quicldy enters into an ester bond with a hylllo~yl group
without further activation. Suitably activated polyamide membranes carry triazine groups,
for inst~nce~ that can react with amino groups of nucleic acid analogs with the forrnation of
a covalent bond. The activation can also take place by means of bifunctional re~g~nte,
squaric acid derivatives of WO 95/15983 or glutaraldehyde (GB 2197720).
The binding of the analogs can also be realized by coating the carrier surface with
nucleotide sequences that are complementary to a part of the sequence of the nucleotide
analogs. The binding of the di~el ell~ nucleic acid analogs to the binding regions can take
place simlllt~neously or sequentially.
Af[er a s--ffiçient amount of time has passed for the binding to take place, it is advantageous
to wash away any nucleic acid analogs ~hat have not bound or are bound in~l-fficiently,
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along with any binding reagents that were used. This is performed preferably under
conditions in which non-bound nucleic acid analogs cannot bind with nucleic acid analogs
that are bound to other regions.
R~ie.~lly, it is also possible to build upon the nucleic acid analogs on the different sites of
the surface by means of monomeric units. The technolog,v described in WO 92/10092 or
WO 90/15070 can be used for this purpose. Appropriate monomers are described in
WO 92/20702, for instance.
Another subject matter of this invention is a method for the sequence-specific detection of a
nucleic acid using the solid carrier provided by this invention.
Detectable nucleic acids are natural or artificial nucleic acids. "Nucleic acids" therefore also
refer to nucleic acid analogs. The nucleic acid to be detected, however, is the RNA or
DNA in particular that is characteristic for an organism cont~inin~ nucleic acids, e.g. a
virus, a b~ct~rillm, a multicellular organism, a plasmid or a genetic condition such as a
predisposition or a disposition for a certain disease or a spontaneous genetic mutation. The
RNA and DNA in this case is basically of genomic origin or an origin derived therefrom. An
important class of nucleic acids in the context of this invention are the results of a nucleic
acid amplification. These results are also referred to as "~mplific~tes" or "amplicons" below.
The nucleic acids can be present in either their raw form or in a purified or processed form.
A purification can also take place by separating the nucleic acids from cell components in a
preparatory step, e.g. an affinity separation step. The nucleic acids can also be enzym~ti~ ~lly
~Yt~nr1erl, specifically amplified or transcribed.
For the sequence-specific detectic~n of nucleic acids, the carrier provided by this invention
has a nucleic acid analog bound to a site that has a base sequence that is complement~ry to
a base sequence of the nucleic acid to be detected This base sequence is selected
intentionally so that it can specifically reveal the presence of the nucleic acid. In the normal
case this means that the mixture contains as few as possible--and preferably no--
additional nucleic acids having the same total sequence. It must be mentioned, however,
that the carrier provided by this invention can also be used to specifically detect groups of
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nucleic acids. It can be a task of the method, for in~t~nf ~ to detect any member of a certain
taxonomic group, e.g. a family of bacteria, by means of its nucleic acids. In this case, the
base sequence of the nucleic acid analog can be intentionally selected so that it lies in a
conserved region but only occurs in members of this taxonomic group.
An additional nucleic acid analog that has a base sequence that is not compl~Pmpnt~ry to the
same base sequence is preferably bound to a di~Te~ L site of the surface. It can be a nucleic
acid analog, the base sequence of which can be shorter or longer, or which can differ from
the first nucleic acid analog by one or more bases. The difference in the base sequence
depends on the task to be solved. The differences can include point mutations, or smaller
dçlçtiom and insertions, for instance. In many genetic tii~e~es, such as cystic fibrosis, the
sequences of the nucleic acid analogs differ in terms of individual positions (point
mllt~tion~) and numerous positions (deletions, for example at ~ 508).
The mutation to be detected is preferably positioned close to the middle of the base
sequence of the analog.
The sequences of the analog can also be intpntic~n~lly selected so that their hybridization
positions differ by one base each, even though the lengths are idPntic~l (overlap). The
sequences can also be intentionally selected so that the hybridization regions are ~ cent to
each other on the nucleic acid to be cletecte~1
Numerous mutations can also be detected in the same or di~elent nucleic acids by sf-lecting
nucleic acid analogs with sequences that are complpm~nt~ry to the sequence on these
nucleic acids.
In an especially easy case in which the purpose is to determine which of two alleles is
present in a complex, two nucleic acid analogs are bound to di~erellL sites of the surface of
the solid carrier, the base sequences of which differ in exactly that position where the alleles
also differ. A nucleic acid analog is therefore selected that is complçm~nt~ry to a certain
sequence of one allele, while the other nucleic acid analog is comrlem~nt~ry to the sequence
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.
of the other allele. The length and hybridization sites of the nucleic acid analogs are
identical.
All alleles are usually detected for cystic fibrosis, for in.~t~nce The wild-type contains two
healthy alleles. Heterozygotes contain one m-lt~ted and one wild-type sequence, and
homozygotic mllt~nt~ contain two mutant nucleic acids. In this case, the intent is not just to
determine if mllt~nts are present, but to determine if it is a heterozygotic or homozygotic
case. In accordance with this invention it is possible to sim~llt~nçously q~ ely detect
both alleles and thereby differentiate between the three cases described.
In many cases, especially in oncology and in the determination of infectious parameters,
mllt~ted cells/particles are usually located in the background of non-m~lt~te~l/normal cells.
In these cases, selective detecti~n can not be performed reliably or at all using methods
provided by the state of the art. The analysis of ras mutations from DNA from stool
samples, for instance, requires that a m--t~ted sequence be reliably detected in the presence
of approx. 100 normal sequences (Science 256, 102-105 (1992)). In the field of infectious
diseases, it would be desirable to det~rmirle different HIV populations in one infected
patient. The quantity of many mnt~nt~: of these HlV pop--l~ti~-n.~ is less than 2% compared
with all HrV sequences, however. The method provided by this invention therefore makes it
especially quite possible to investigate mixtures of nucleic acids that are very similar to each
other, even if one of the nucleic acids is present in a much greater quantity than the nucleic
acid to be determined.
The lengths of the bound nucleic acid analogs are preferably identical. An applop,iate length
has proven to be between 10 and 100, and preferably between 10 and 50 bases. Especially
good results are obtained with nucleic acid analogs that are between 10 and 25 bases long.
To perform the method provided by this invention, the sample c~ nucleic acid is
brought in contact with the sites on the surface of the carrier that have bound the nucleic
acid analogs. This can be performed, for in~t~nce7 by bringing the solid carrier into the
sample fluid or pouring the sample fluid onto the solid carrier in one or more portions. The
nucleic acids in the sample fluid can be denatured (single-strand) before they are brought in
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11
contact with the carrier. A major advantage of the invention, however, is the fact that a
prelimin~ry denaturation step can be çiimin~te~1, e.g. by using PNAs. The PNAs force a
strand out of the double-stranded nucleic acid to be determinp~rl The only important
requirement is that the sample be brought in contact with the solid carrier under con~litione
in which the nucleic acid to be ~letected binds spe~ifie~lly to the appropriate site on the
surface by means of the nucleic acid analog which is compl~mPnt~ry to one sequence of the
nucleic acid to be determined. These conditions can be ~ for ~li~elel~ types of
nucleic acid analogs, of course, but they are easily determined for given nucleic acid analogs
by pelr~ ning tests. In the normal case, these con-litions are based on the conditions that
are known for carriers loaded with oligonucleotides. If nucleic acid analogs are used as
described in WO 92/20702, however, con~ ione can be selected that are much di~ele-.l
from the hybricli~tion conditions for the corresponding oligonucleotides. It has proven to
be appropriate, however, to use much less salt than when the corresponding
oli~oml~l~otides are used. For instance, the presence of less than 100 mM and, more
preferably, less than 50 mM, and most preferably, less than 10 rnM salt is recommPn~ed
Under these conditions, it would not be possible to s~lffi~iently di~lel-liate between nucleic
acids having similar sequences using oligonucleotides having the same sequence.
The sample is kept in contact with the surface as long as necessary to achieve a s .ffi~.ient
binding of the nucleic acids to the appropriate site on the surface. This period is usually a
few mimltes
In the next step, it is determined whether the nucleic acid has bound to the surface and, if
so, to which site. This is considered an in~1ic~tion of the presence of a nucleic acid that
contains a base sequence that is compl~omPnt~ry to the nucleic acid analog bound to this site.
The binding can be determined using various methods.
Instruments are already available with which changes on specific sites of surfaces can be
determined directly. For methods that use these types of instruments it is not even necesc~ry
to remove the sample cont~ining the nucleic acid from the surface after it has been applied.
Normally, however, it is preferable to remove the fiuid from the surface and use a wash
solution to remove any . ~ .g reagent that is still adhered to the surface. This step
.
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provides the advantage of also washing away sample components that can interfere with the
determin~tion of the binding
In a prerel~ ed embodiment, the binding of the nucleic acid to be detected with the nucleic
acid analog is determined by means of a label that is inserted in the nucleic acid to be
determined in a step that is performed before the sample is brought in contact with the
sl-Tf~ee The label can be a detect~ble group such as a fluorescent group, for in~t~nce. This
determination can be performed optically using a microscope or in a measuring cell
provided for this purpose. While the site at which the binding took place is an indication of
the presence of a nucleic acid having a certain sequence, the quantity of label at a
predetermined site can be used as an indication of the quantity of the nucleic acid to be
determined.
In an especially p,erel.~d embodiment, the nucleic acids to be detected are the products of a
nucleic acid amplification method such as the polymerase chain reaction as described in
EP-B-0 202 362, or NASBA as described in EP-A-0 329 822. It is important that the
nucleic acid sequence to bind with the nucleic acid analog be amplified by the amplification
method. The better the amplification method m~int~ine the original sequence--that is~ the
fewer errors that are incorporated into the sequence during amplification--the more
suitable the amplification method. The polymerase chain reaction has proven to be
especially suitable. The amplification primers are selected specifically so that the nucleic
acid sequence to be detected lies in the region between the hybridization sites.
It has also proven advantageous to insert the label required for the detectinn reaction into
the amplificate during amplific~tion This can be performed, for in~t~nr~ by using labelled
primers or labelled monon-lcleoside triphosphates.
The binding that took place can be detected directly without inserting a label, for in~t~nce,
by using an interc~l~ting agent. These agents have the characteristic of depositing selectively
on double-stranded compounds that contain bases, inr,~ lin~ the complex of the nucleic acid
analog and the nucleic acid bound in sequence-specific fashion. The presence of the
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complexes can be detected using specific characteristics of intercalating agents, e.g.
fluorescence. Fthi~ m bromide is an especially suitable agent.
Another method for detectin~ hybrids without inserting a label is based on surface plasmon
resonance, as described in EP-A-0 618 441, for in~t~n~e.
According to another possible method ~or determinin~ the bin(lin~;~ the surface is brought in
contact with a solution of an antibody labelled for detection. This antibody is directed
against the complex con~isting of the nucleic acid analog and the nucleic acid to be
determined. Antibodies of this nature are described in WO 95/17430, for in~t~nce
The detection of the hybrids depends on the type of labelling used. The hybrids can be
detected with a scanner, a CCD camera or a microscope, for in~t~nce
This invention provides numerous advantages. In particular, it allows detecting sequence
differences in nucleic acid regions located within secon-l~ry structures. With this invention it
is also possible to increase the sensitivity of detection> because it can use a greater absolute
quantity of bound nucleic acid to be determined than traditional methods. It is also possible,
in particular, to increase the signal-to-noise ratio compared with methods that use
oli~on~lcleotides. In the first attempt, a signal-to-noise ratio of less than 1: 1000 was
obtained.
The invention can be used in at least two fields. In the first case, the solid carrier is used to
detect known mutations and polymorphisms. In this case, the number of mllt~tion.~ and
polymorphisms to be determined is an indicator of the number of ~lirre~ nucleic acid
analogs or sites required on the surface. The sequence of the nucleic acid analogs is
specially coordinated with the sequence of the nucleic acids around the mutations and
polyrnorphisms. Preferably, the sequences are selected in such a way that the base by which
nucleic acid analogs having similar sequences differ is located in or near the middle of the
sequence.
The carriers provided by this invention can be used in the following fields:
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14
I~Lfectious (li~e~ses, the simlllt~neous det~ n of different analytes/parameters, and in
investig~ti~ns of the condition of a gene in a b~ct~rillm or virus, e.g. for multi-drug
rP~ict~nr.e studies.
Oncology (detection of mutations in tumor suppressor genes and oncogenes, and in the
det~rrnin~tion of the relationship between mllt~ted and normal cells).
Investigation of inherited ~i~e~es (cystic fibrosis, sickle cell anemia, etc.).
Tissue and bone marrow typing (MHC complex) (see Clin. Chem. 41/4, 553-5 (1995)).
In a second potential application, a sequence of short nucleic acid fr~mentc can be
determined using the méthod called "sequencing by hybridization". In this method, the same
number of different nucleic acid analogs are immobilized as there are pelllluLalions of the
s~lected length of the sequence. To achieve a sufficient level of sensitivity, 4N sites are
required, with N equal to the number of bases in each nucleic acid analog. Preferably, N is
between 5 and 12. Correspondingly fewer sites are required to sequence very short DNA
fr~gment~ The method for sequencing unknown nucleic acids using the "sequencing by
hybri~li7~tion" method is described in WO 92/10588.
An advantage of this invention is the ~act that the specificity of the hybridization is largely
independent of the contlitic~n~ in the sample. This f~cilit~t~ ~imlllt~neous binding of nucleic
acids to different regions on the surface.
Surprisingly, it was shown that nucleic acid analogs such as PNA have an Pxc~ nt ability to
discriminate between sequences on the surface. This disc~ n was better than was to
be expected from the melting temperatures of analogous, dissolved compounds.
Surprisingly, the carriers provided by this invention are even suitable for use in numerous,
consecutive det~rmin~tions of nucleic acids. After a determination is performed, the carrier
that is in contact with a fluid undergoes heat tre~tm~nt A temperature is selected at which
43001~NGLDOC
CA 02214430 1997-09-02
the bond between the nucleic acid analog and the nucleic acid is dissolved. The carrier is
then available to perform another dete~ ;Qn With the method provided by this
invention, it is possible for the first time to determine relative q~l~nti~ies of very similar
nucleic acids located next to each other in a sample in a concentration range of at least two
logs. It has been possible to q~l~ntit~tively dele~ .e ~ using seq l~neing methods, for
instance. The m~imllm level of discrimin~tion available with this method was l: l0,
however.
With the method provided by this invention it is also possible to bind double-stranded DNA
to the immobilized nucleic acid analogs of the solid carrier without a denaturation step.
Comparison studies have shown that this is not possible with immobilized DNA. It has also
been shown that mi~m~tches that are not located in the center of the hybrid can also be
~listin~ hed with a high degree of selectivity.
The sequences of the PNA molecules are shown in Fig. la. They are used as examples to
explain the method. The PNAs were prepared as described in WO 92/20702.
Fig. lb shows the sequences of the DNA molecules that are homologous to the PNA
sequences from Fig. la and that were used for the DN~/ODN hybridization experiments.
Fig. 1c shows the sequences of the complementary oligom~cleotides (ODN) used that were
labelled with digoxigenin on the S'-phosphate end using the S'-DIG End Labelling Kit
~13Oehringer l~nnheim) and that were phosphorylated with polynucleotide kinase and 32p_
g-ATP 5'.
Fig. ld shows the feasible combinations of oligonucleotide (ODN) and PNA for forming
hybrids. Td~.ntic~l col~hil1~tions apply for hybri~li7~ti~ ns between DNA probes (DNA, see
Pig. lb) and oligonucleotides (ODN, see Fig. lc).
Fig. 2 shows the hybridization results from Exarnple 4 to illustrate the selectivity of the
method. The conditions were: 200 nl spot volume (l00; l0; l; 0. l mM PNA, one
concentration per cleavage), incubation at 45 ~C.
4300E~NGLDOC
CA 022l4430 l997-09-02
16
Fig. 3 shows the hybritli7~tic)n results from Exarnple 6. They verify that PCR amplicons are
detected by immobilized PNA probes. The con~litionc were: 200 nl spot volume (100; 10; 1;
0.1 mM PNA, one concentration per cleavage), incubation at 45 ~C. The labels in Fig. 3
mean:
Control experiment, ODN la (lpMol, lnM), line I (PNA 1), line 2 (PNA 2)
line 3 ~PNA 3)
II ss Amplificate (118 bp, one-fold DIG-labelled)
5 min heat denaturation at 94 ~C
(50 ml PCR preparation diluted in 1 ml hybridization buffer)
Line 1 (PNA 1), line 2 (PNA 2), line 3 CPNA 3)
m ds Amplificate (118 bp, one-fold DIG-labelled)
(50 ml PCR preparation diluted directly in 1 ml hybridization buffer)
Line 1 OENA 1), line 2 ~PNA 2), line 3 (PNA 3)
Fig. 4 shows the results of the qualitative and q ~~ntit~tive analysis of analyte mixtures by
means of PNA arrays. The conditions were: 200 nl spot volume (100 rnM PNA, one PNA
per line, one analyte mixture per cleavage).
Fig. 5 shows the effect of linker length on the hybridization. The con~itit~ns were:
1 rnl spot volume (100; 40; 20; 10; 5; 1 mM PNA, one concentration per cleavage, (Ado)3-
PNA in row 1, (Ado)6-PNA in row 2, (Ado)g-PNA in row 3). The labels in Fig. 5 mean:
A. Prehybridization / hybridization in S mM sodium phosphate, 0.1% SDS, pH 7.0
B. Prehybridization / hybridization in 10 mM sodium phosphate, 0.1% SDS, pH 7.0
C. Prehybridization / hybridi7~ltion in 25 mM sodium phosphate, 0.1% SDS, pH 7.0
Figures 6a - 6c d~mon~rate that PNA-derivatized membranes can be used many times aflcer
regeneration. The con~litic)ns were: 1 ml spot volume (100; 40; 20; 10; 5; 1 mM PNA, one
concentration per cleavage).
43~
CA 022l4430 l997-09-02
17
The labels in Fig. 6 mean:
6a: Signal intçn~i~ies after the first hybritli7~ti~n
6b: Signal intçn.~ities after the regeneration procedure
Membrane 1: No regeneration (controls)
~embrane 2: Regeneration with 0.1 M sodium hydroxide solution, RT 1 h,
2 x 10 min hicli~tilled water RT
~embrane 3: Regeneration with 1 M sodium hydroxide solution, RT, 1 h, 2 x 10 min bidistilled water RT
~embrane 4: Regeneration with ~ tilled water, 70 ~C 1 h, 2 x 10 min bidistilled
water RT
~embrane 5: Regeneration with 0.1 M sodium hydroxide solution, 70 ~C 1 h,
2 x 10 min bidistilled water RT
6c: Signal intçn~ities after rehybrirli7~tion
~his invention is explained in fu~ther detail using the following examples:
4300ENGL.DOC
CA 02214430 1997-09-02
18
Examples
General:
The nucleic acid analogs used were m~mlfact lred as described in WO 92/20702. Unless
indicated otherwise, chçmie~l~ and reagents were products of Boehringer ~annhP.im
GmbH.
Example l:
Covalent Derivatization of Nylon Membranes
200 nl of a solution that contains PNA in the desired concentration in 0.5 M sodium
carbonate pH 9.0 are applied to an Tmnlllnodyne ABC membrane (Pall) with a pipette. After
the spots are dry, the membrane is washed with 0. l M sodium hydroxide solution to
deactivate any reactive functional surface groups that may still be present. The membrane is
washed a second time with water and then dried.
Example 2:
Detection of a Hybri~li7ation Event Using L ~minesc~nce
The membrane is derivatized as described in Example l using lO0 IlM, lO mM, 1 IlM and
0.1 mM PNA solutions. It is then prehybridized in a 50 ml hybridization vessel with lO ml
hybri(li7~tion buffer (lO mM sodium phosphate, pH 7.2, 0.1% SDS (sodium
dodecylsulfate)) in a hybridization oven at 45 ~C. After 30 mimltç~, lO ml of a solution that
contains the DIG-labelled oligonucleotide in a 1 mM concentration is added and the
complex is hybridized for another 60 min~ltes It is then washed for 2 x lO mimltes with 25
ml wash buffer each time (5 mM sodium phosphate pH 7.2, 0.05% SDS) at 45 ~C. Thedetection reaction is performed according to the protocol for digoxigenin detection (DIG
Detection Kit, Boehringer l~annh~im GmbH, BRD). The anti-DIG-AP conjugate is used in
430t~NGLDOC
-
CA 022l4430 l997-09-02
19
a 1:10000 dilution. CDP-StarTM is used in a 1:10000 dilution as the substrate for the alkaline
phosphatase.
Example 3:
Detection of a Hybri~1i7~tion Event Using Fluorescence
The membrane is derivatized as described in Example 1 using 100 ,uM, 10 ,uM and 1 ~M
PNA solution. The membrane is prehybridized in a 50 ml screw-top container with 10 ml
hybridization buffer (see Example 2) in the oven at 45 ~C. A~er 30 min-ltç~, 10 m1 of a
solution that contains a fluorescent-labelled oligonucleotide in a concentration of 1 IlM is
added, and the preparation is hybridized for another 60 mimltes The membrane is then
washed for 2 x 10 mimltes with 25 ml wash buffer each time (see Example 2) at 45 ~C. The
membrane is dried, then the intensity of the fluorescence is measured.
Example 4:
Selectivity of the Method
Three membrane strips are derivatized with three (Ado)6-PNA molecules each that differ
according to one or two positions of their base sequence (see Fig. la, SEQ.ID.NOS. 1, 2,
3), using PNA solutions in a concentration range of between 100 mM and 0.1 mM asdescribed in Example 1. The membrane strips are prehybridized with 10 ml hybridi7~tinn
buffer for 30 min~ltes in 50 ml screw-top cont~in~rs In the next step, one of the three DIG-
labelled oligonucleotides (Fig. lb, SEQ.ID.NOS. 4, 5, 6) is added. After hybridizing for 60
mimlte~, the membranes are washed for 2 x 10 mimltes with 25 ml wash buffer each time.
The hybridization events are detecte~l as described in Example 2.
All possible double-stranded hybrids between the PNA molecules involved and the
oligonucleotides are shown in Fig. ld. Figure 2 illustrates that, in almost every case, the
only oligonucleotide detected is the one that is exactly complçm~nt~ry to the immobilized
nucleic acid analog (PNA 1, PNA 2, PNA 3). The signal-to-noise ratios (S/N) can also be
4300ENGL.DOC
CA 02214430 1997-09-02
estim~ted from the figure. They were evaluated q~l~ntit~tively, and the results are presented
in Table 1.
Table 1
Hybrid (PNA/ODN) S/NSignal (Hybrid)/Signal ~Match)
1/1 655.2 100.0%
2/1 20.7 3.2%
3/1 10.3 1.6%
1/2 23 . 1 2.6%
2/2 871.8 100.0%
3/2 6.4 0.7%
1/3 109.4 22.7%
2/3 12.3 2.5%
3/3 481.1 100.0%
Example 5:
Q~l~ntific~tiQn
Membrane strips are derivatized with three (Ado)6-PNA molecules with different base
sequences (Fig. la, SEQ.ID.NOS. 1, 2, 3) in a concentration of 100 mM as described in
Fx~mrle 1. They are then prehybridized in 20 ml hybridization vessels with 10 mlhybridization buffer (see Example 2) at 45 ~C. The buffer is replaced after 30 mimltes In
eXpPrim~nt~ 1 through 7, the buffer to be added differs according to the analyteconcentrations of the DIG-labelled components - oligonucleotide 1, 2 and 3, SEQ.ID.NOS.
4, 5, 6. The strips are hybridized for 60 mimltes at 45 ~C and then washed for 2 x 10
mimltes with 10 ml wash buffer. The detection is peTformed using the procedure described
in F.x~mrle 2. The Illminescçnce signal is recorded with a l~lminçsc~nce imager and then
evaluated (Fig. 4).
~3c~.~rTT no~
CA 02214430 1997-09-02
21
The signal intçn~ities found can be used to reach a qualitative or semi-q l~ntit~tive finding
regarding the composition of the analyte complex. Absolutely qu~ntit~tive finflin~ can be
reached after the signal in~Pn~iti~e are calibrated.
Example 6:
Detec~ion of PCRAmplicons
A. Obtaining a Suitable Analyte (Amplificate)
A double-stranded DNA fragment is ligated in a pUCl9 plasrnid, the sequence of which is
complem~nt~ry to the PNA probe PNA 1. The plasmid is transformed in E. coli, cloned, and
then sequenced. For the subsequent hybridization experiments, a section of the plasmid
sequence is amplified and DIG-labelled during the amplification reaction. The amplification
is performed in a total volume of 50 Ill. The amplification complex consists of 1 ,ul plasmid
(1 ng/~ l primer Fl (10 ,uM~, 1 ,ul DIG primer Rl (10 ll~, 5 ,ul 10 x PCR buffer
(100 mM Tris/HCl, 15 mM MgCl2, 500 mM KCI, pH 8.3), 2 ~l dNTP solution (10 mM
dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP in distilled water, pH 7.0), 0.5 ~l Taqpolymerase (5 units/~ll) and 38.5 ml water.
Primer Fl: 5'-GTA AAA CGA CGG CCA GT-3' (SEQ.ID.NO. 12)
Primer R1: 5'-DIG-AAC AGC TAT GAC CAT GA-3' (SEQ.ID.NO. 13)
Each reaction mixture is warmed to 96 ~C for 3 mimltes In the next step, 30 rounds of a 3-
level PCR cycle are performed (45 sec. 96 ~C, 30 sec, 48 ~C, 1 min 72 ~C). In the last cycle,
the elongation step is increased by 5 mimltes at 72 ~C.
B. Hybridization Reaction
The membranes are derivatized with three (Ado)6-PNA sequences each that differ according
to ome or two positions in their base sequence (see Fig. la, SEQ.ID.NOS. 1, 2, 3) using
~13~N~'TT T~9C
CA 02214430 1997-09-02
22
PNA solutions in a concentration range between 100 ,uM and 0.1 ,uM as described in
Example 1. The membrane is p~ ealed in a 20 ml hybridization vessel with 5 ml
hybridization buffer at 45 ~C. The buffer is replaced after 30 rnim-tes and the analyte
solution is added. To make the analyte solution, the amplification complex is diluted directly
(ds a-m--plicon) and, after 5 mimltcs of heat denaturation (ss arnplicon), in 1 m1 hybridi7~tion
buffer. Ai~cer hybridization for 1 h, 2h 30 min and 4 h at 45 ~C, the membranes are washed
for 2 x 10 minlltes with 5 ml wash buffer each time. Hybri~1i7~tion events are c~etected as
described in Exarnple 2 ~Fig. 3).
Nine fields are shown in Figure 3. The difference between each row is the incubation period
(4 h, 2-1/2 h, and 1 h). The difference between each column is the type of nucleic acid to be
detected Three overlapping rows of spots are applied to each of the 3 fields of column I.
The difference between the rows is the sequence of the PNAs, while the difference between
the columns of each field is the concentration. The specificity and the ability to be qll~ntified
are in~lic~ted in column I for the case in which oligonucleotides are used as the detecting
nucleic acid.
The figure illustrates the infl~nce of inc~b~tiQn time. It is clear that an excellent sequence
di~ rimin~tion for ODN l a and the amplificates is obtained after hybridization for just one
hour. The difference between columns II and III in Fig. 3 is that an amplificate that was
previously made single-stranded is used in one case as the nucleic acid to be detected In
column III, an amplificate that was not previously made single-stranded is used as the
nucleic acid to be detected The signals in~lic~te clearly that it is not necessary to denature
double-stranded nucleic acids before applying them to the solid carrier. This decreases the
number of working steps (heating step, single strand separation, wash step) and, therefore,
reduces the danger of cont~min~iQn. PNA probes in combination with low-salt conditions
therefore offer clear advantages over DNA probes.
430aENG~DOC
CA 02214430 1997-09-02
23
Example 7
Comparison of PNA / DNA Hyhri~li7~tion
Membrane strips are del;v~lized with three (Ado)6-PNA sequences each (SEQ.ID.NOS. 1,
2, 3) and three DNA molecules each (SEQ.ID.NOS. 8, 10, 11) that differ according to one
or two positions in their base sequence (see Fig. la and lb) using 50 mM solutions as
described in Fx~mple 1. Unlike Example 1, the spot volume is 400 nl instead of 200 nl. The
membrane strips are prehybridized in 20 ml hybricli7~ti~n vessels with either 5 ml low-salt
buffer (see Example 2) or high-salt buffer (6 x SSC; 0.9 M NaCl, 90 mM sodium citrate,
0.1% SDS, pH 7.0) at either 37 ~C or 45 ~C for 30 min~-tes In the next step, one ofthe
three DIG-labelled oligomlrleotides ~Fig. lc, SEQ.ID.NOS. 4, 5, 6) is added. After a
hyhri~li7~tion step of 60 min~lte~, the strips are washed for 2 x 10 min-ltes with 5 ml wash
buffer each time at 37 ~C or 45 ~C. The wash buffer from Example 2 is used for the low-salt
experiments. For the high-salt experiments, a 1 x SSC buffer with 0.02% SDS, pH 7.0 is
used. The hybridization results are detected as described in Example 2. The eva~ ti~n is
performed q~ntit~tively and is illustrated in Table 2.
Both the DNA and PNA probes are able to completely disc~ a~e between
compl~mlont~ry, single-stranded target sequences of single and double mi~m~tchedsequences. PNA probes demonstrate clear advantages over the DNA probes for certain
types of mi~m~trhe~, especially when they are not located in the middle of the sequence, but
rather shifted to the end. This becomes especially clear in the r~mrle of a decentral G/T
mi~m~trh (probe 1 / ODN 3), which is tolerated by the DNA probe much more strongly
than by the PNA probe having the identical sequence.
430~ENGLDOC
CA 02214430 1997-09-02
24
Table 2
~ ,~," s: ~s ~33~ 3~,S, ~,3 ~ 7- ~ P~r ~s
ODN 1 PNA45 ~C, low salt 100.0% 1.2% .5~
DNA 37 ~C, high 100.0% 1.2% 6.4%
salt
ODN 2 PNA 45 ~C, low salt 1.1% 100.0% 2.9%
DNA 37 ~C, higlî < 2%* 100.0% < 2%*
salt
ODN 3 PNA 45 ~C, low salt 26.0% 1.5% 100.0%
DNA 37 ~C, high 66.5% < 2%* 100.0%
salt
* A more exact value cannot be detennined because the spot intensity is lower than the
standard deviation of the background signal.
~13nn~1~Tr.T T70C
CA 02214430 1997-09-02
Example 8
TnflllP.n- e of the Length of the Linker Between the Membrane and PNA Probes
Membrane strips are derivatized with PNA molecules (see Fig. la, SEQ.ID.NOS. 7, 1, 9)
that differ according to the length of the linker (Ado3, Ado6 or Ado9) using PNA solutions
in the concentration range between 100 ~lM and 1 ~lM as described in Example 1. Unlike
Example 1, the spot volume is 1 111 instead of 200 nl. The membrane strips are
pl~hyl~lidized in hybril1i7~tion cont~iners with 10 ml hybricii7~tion buffer (5, 10 or 25 rnM
sodium phosphate, 0.1% SDS, pH 7.0) for 30 min~ltes at 35 ~C. In the next step, 10 pMol
32P-labelled oligonucleotide (see Fig. lc: ODN lb, SEQ.ID.NO. 4) is added and the
preparation is hybridized for 60 min~ltes at 50 ~C. The membranes are washed for 2 x 10
minlltes with 50 ml wash buffer (5 mM[ sodium phosphate, 0.1% SDS, pH 7.0) at 50 ~C.
The hybridi7~tion events are detected using autoradiography (Fig. 5). The figure shows that
a longer linker greatly improves the hybridization.
Example 9
Reuse of PNA Membranes
A membrane is derivatized with (Ado)6-PNA molecules (see Fig. 1 a: PNA lb, SEQ.ID.NO.
1) using PNA solutions in a concentration range between 100 ~lM and 1 ,uM as described in
Example 1. The PNA is applied in five identical concentration sequences. The spot volume
is 1 ,ul, as in Example 8. The membrane is plehyl .idized in a hybridi:zation vessel with 10 m1
hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 mimltes at 35
~C. In the next step, 10 pMol 32P-labelled oligonucleotide (see Fig. lc: ODN lb,SEQ.ID.NO. 4) is added and the prepalalion is hybridized for 60 minllt~i at 50 ~C. The
membrane is washed for 2 x 10 mimltes with 50 ml wash buffer (5 mM sodium phosphate,
0.1% SDS, pH 7.0) at 50 ~C. The hybri~i7~tic)n events are detected using autoradiography
(Fig. 6a). AP~er the autoradiography is performed, the membrane is cut into five iclenti
strips. These membrane strips are each treated di~el elllly in the rest of the experiment.
43WENGL.DOC
CA 02214430 1997-09-02
26
Membrane 1 is not inr~lb~ted and serves as the control membrane. Membrane 2 is incub~ted
for 60 min-ltcs at room temperature with 50 ml 0.1 M sodium hydroxide solution.
Membrane 3 is inc~lbated for 60 mimltes as well, with 50 ml 1 M sodium hydroxidesolution. Membrane 4 is inc~lb~ted for 60 minll~eS at 70 ~C with 50 ml distilled water.
Membrane 5 is inr,~lb~ted for 60 min~tes at 70 ~C with 50 ml 0.1 N sodium hydroxide
solution. All membranes are then washed with tli.~tilled water for 2 x 10 minlltes After this
procedure is ct)mrlete~l, autoradiography is pP.rfoTmed once more (Fig. 6b). These
membrane strips are then used a second time in a hybridization reaction as described, and
the hybridization events are detected using autoradiography (Fig. 6c).
As shown in Fig. 6b, the difre~ tre~tment methods yield very di~le..~ results. Tre~tmPnt
with bidistilled water at 70 ~C (membrane 4) causes the membrane to regenerate almost
completely. An unexpected discovery was the fact that the success of the regeneration is
poorer if conditions are used that are common for the denaturation of nucleic acids. As
such, the incub~tion of membrane 3 with 1 M sodium hydroxide solution at room
temperature yields virtually no regeneration effect. Decreasing the concentration of sodium
hydroxide solution from 1 M to 0.1 ~ increases the degree of regeneration at room
temperature (membrane 2) and at 70 ~C (membrane 5). None of these conditions, however,
results in an even slightly good degree of regeneration, as is the case with bi~ tillecl water
(membrane 4). The example shows that these conditions are important parameters for the
~ffici~nt denaturation of membrane-bound PNA/DNA double-strands.
Regardless of the regeneration method, all membranes can be reused for hybridization
(Fig. 6c), without considerably worsening the signal-to-noise ratio. Up to 6 rehybridizations
could be performed without a noticeable effect on the PNA membranes' ability to
regenerate or rehybridize.
43001~NGL.DOC
CA 02214430 1997-09-02
SEQUENCE LIST~G
(1) GENERAL ~FORMATION:
(i) APPLICANT:
(A) NAME: Boehringer ~nnh~im GrnbH
(B) STREET: S~n~lhoferstr~ 116
(C) CITY: M~nnh~im
(E) COUNTRY: DE
(F) POSTAL CODE (Zn?): 68298
(G) TEI~PHONE: 0621 759 4348
(EI) TELEFAX: 0621 759 4457
(ii) TITLE OF ~IVENTION: Sequence-Specific Detection of Nucleic Acids
(iii) NUMBER OF SEQUENCES: 13
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC comp~til~le
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) sTRANDFnNEss: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAMEfKEY: Modified-site
(B) LOCATION:15
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:14
43001~NGLDOC
CA 02214430 1997-09-02
,
28
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-~hyll~illyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME/KEY: Modified-site
~3) LOCATION:13
(D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -cytosyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
(ix) F~ATllRE:
(A) NA~JKEY: Modified-site
(13) LOCATION:11..12
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:10
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:9
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-(( 1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATI~RE:
(A) NAME/KEY: Modified-site
~3) LOCATION:8
~D) OTEIER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site
(B) LOCATION:7
(D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -thyminyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
4300E~GL.DOC
CA 02214430 1997-09-02
29
(B) LOCATION:6
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -guaninyl)acetyl)-N-(z-~minoettlyl)-beta-alanine
(ix) ~EATI~
(A) NAMEtKEY: Modified-site
Q3) LOCATION:5
(D) OTE~R INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATI~E:
(A) NAMElKEY: Modified-site
(13) LOCATION:4
(D) OTHER lNFORMATION:/product= "OT~R"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:3
(D) OTHER INFORMATION:/product= "OTE~ER"
/note= "Xaa is
N-((l-Lhy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:2
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -guaninyl)acetyl)-N-(~-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAMEIKEY: Modified-site
~B) LOCATION: 1
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3 ,6-dioxa-oc
tano-l-yl)-ethyl)-beta-alanine"
.
(lX) FEATllRE:
(A) NAMEIKEY: Modified-site
Q3) LOCATION:16
~D) Ol~RINFORMATION:/product= "Ol~R"
/note= "Amide"
4300ENGLDOC
.~
CA 02214430 1997-09-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
(2) INFORMATION FOR SEQ ID NO: 2:
Cl) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECUIE TYPE: peptide
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
Q3) LOCATION:15
(D) OTHER~FORMATION:/product= "OTHER"
/note= "Xaa is
N~ adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
~ (A) NAME/KEY: Modified-site
~13) LOCATION: 14
(D) OTHER ~FORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-thy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION: 13
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:11..12
~D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
4300ENGLDOC
CA 02214430 1997-09-02
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:10
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NA~/K~Y: Modified-site
(B) LOCATION:9
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:8
(D) OTEIER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:7
(D) O l~R ~IFORMATION:/product= "OT~R"
/note= "Xaa is
N-((l -thyminyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:6
(:D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) N~¢/KEY: Modified-site
(B) LOCATION:5
(D) Ol~RINFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-~l-adeninyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
430~1ENGLDOC
CA 02214430 1997-09-02
32
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:3
Q~) OTHER INFO~MATION:/product= "OTHER"
/note= "Xaa is
N-((l-ll~y~ lyl)acetyl)-N-(2-~min~ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~B) LOCATION:2
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:/producP "OT~R"
/note= "Xaa is
N-((l -thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3 ,6-dioxa-oc
tano- 1 -yl)-ethyl)-beta-alanine"
(ix) FEATllRE:
(A) NAME/KEY: Modified-site
~B) LOCATION: 16
- (D) OTHER INFORMATION:/product= "OTHER"
/note= "Amide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
430()ENGL.DOC
CA 02214430 1997-09-02
x) FEATI~
(A) NAME/KEY: Modified-site
~B) LOCATION:15
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-~minoethyl)-beta-alanine"
~lX) ~ATInRE:
(A) NAME/KEY: Modified-site
~13) LOCATION: 14
~D) OTE~R INFORMATION:tproduct= "OTHER"
/note= "Xaa is
N-((1-LLy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:13
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) ~EATURE:
(A) NA~/KEY: Modified-site
~B) LOCATION: 11 . .12
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) ~EATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:10
(D) Ol~RINFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:9
(D) Ol~R INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) FEATI~E:
(A) NAME/KEY: Modified-site
(13) LOCATION:8
(D) Ol~R~FORMATION:/product= "OTHER"
/note= "Xaa is
43~;Nt'.T. T'W
CA 02214430 1997-09-02
34
N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/~Y: Modified-site
(B) LOCATION:7
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-~hymll~yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~13) LOCATION:6
(D) OTHER INl~ORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site
(13) LOCATION:5
~D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:4
(D) OTHER I~lFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATllRE:
(A) NAME/KEY: Modified-site
(13) LOCATION:3
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -~Lylllillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMElKEY: Modified-site
(B) LOCATION:2
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
~D) OTHER INFORMATION:/product= "OTHER"
4300ENGLDOC
CA 02214430 1997-09-02
/note= "Xaa is
N-((l-thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3,6-dioxa-oc
- tano-l-yl)-ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~13) LOCATION:16
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Amid"
.
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
(2)
SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION:15
(D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with
digoxigenin via aminolinker (Boehringer ~nnh~im GmbX BRD)
or 32-P"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
TAGTTGTGAC GTACA 15
(2) lNFORMATION FOR SEO ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs
4300ENGL.DOC-
CA 02214430 1997-09-02
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:~
(A) NAME/KEY: rnisc_feature
(B) LOCATION:15
(D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with
digoxigenin via aminolinker (Boehringer ~nnheim GmbX BRD)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TAGTTGTCAC GTACA 15
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs
~13) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION:15
(D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with
oxigf~nin via arninolinker (Boehringer ~nnhPim GmbX BRD)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TAGTTGTGAT GTACA 15
(2) INFORMATION FOR SEQ ID NO: 7:
430(ENGI~DOC
CA 02214430 1997-09-02
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
~13) TYPE: amino acid
(C) STRANDEDNESS: Single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(ix) ~EATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:15
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:14
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -lhyll~illyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~3) LOCATION: 11..12
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
Q3) LOCATION: 10
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
4~ t'.T T10~
CA 02214430 1997-09-02
38
(B) LOCATION:9
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:8
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-cytosyl)acetyl)-N-(2 -aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:7
~) OTHER INFORMATIO~:/product= "OTHER"
/note= "Xaa is
N-((l -thyrninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:6
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(IX) FEAllJlRE:
(A) NAME/KEY: Modified-site
(B) LOCATION:5
(D) OTHERlNFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:4
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-(( 1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~13) LOCATION:3
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-~llyll~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
430QI~NGLDOC
CA 02214430 1997-09-02
(A) NAME/KEY: Modified-site
(B) LOCATION:2
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alar~ine"
(ix) FEATURE:
(A) NAME/K~Y: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:/product= "OT~R"
/note= "Xaa is
N-~l-~ymillyl)acetyl)-N-(2-amino-N'-(tri(8-amino-3 ,6-dioxa-oct
ano-1-yl)-ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site
(B) LOCATION: 16
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Amide"
(xi) SEQUENCE DESCR~PTION: SEQ ID NO: 7:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs
(13) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
1111111111 llllllGTACGTCACAACTA 30
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
430al3NGL.DOG
CA 02214430 1997-09-02
(A) LENGTH: 16 Amino acids
(}3) TYPE: amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOT~TICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION:15
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-~hylnillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-(( 1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NA~3/~Y: Modified-site
~13) LOCATION: 11 . .12
(D) OTHER ~FORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME~KEY: Modified-site
~B) LOCATION: 10
(D) OTHER INFO:~MATION:/product--"OTHER"
/note= "Xaa is
N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAMEIKEY: Modified-site
(B) LOCATION:9
430~XENGI,DOC
-
CA 02214430 1997-09-02
41
(I)) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATVRE:
(A) NAME/KEY: Modified-site
(B) LOCATION:8
(D) OTHER INFORMATION:/producP "OTHER"
/note= "Xaa is
N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME~KEY: Modified-site
(E3) LOCATION:7
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-(( 1 -~11yl~lillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
~13) LOCATION:6
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1-guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEAT~RE:
(A) NAMEIKEY: Modified-site
(13) LOCATION:S
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/~EY: Modified-site
(13) LOCATION:4
(D) OTHER ~IFORMATION:/product= "OTEIER"
/note= "Xaa is
N-(( l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME/KEY: Modified-site
(13) LOCATION:3
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-~l -lhymi~yl)acetyl)-N-(2-aminoethyl)-beta-alanine~
(ix) FEATVRE:
(A) NAME~KEY: Modified-site
430~.Nr.r. noC
CA 02214430 1997-09-02
42
(B) LOCATION:2
(D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is
N-((1 -guaninyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
(~x) FEATURE:
(A) NAME~KEY: Modified-site
~B) LOCATION: 1
~1)) OTHER INFORMATION:/producP "OTHER"
/note= "Xaa is
N-((l-thyminyl)acetyl)-N-(2-amino-Nl-(nona(8-amino-3,6-dioxa-oc
tano-1-yl)-ethyl)-beta-alanine"
(ix) FEATI~RE:
(A) NAME/KEY: Modified-site
(B) LOCATION:16
(D) OTHERINFORMATION:/product= "OTHER"
/note= "Arnide"
(x~) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
(2) rNFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
~ (ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
1111111111 llllllGTACGTGACAACTA 30
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs
4300ENGL.DOC
CA 02214430 1997-09-02
43
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) S}~QUENCE DESCRIPTION: SEQ ID NO: 11:
l l l l l l l l l L l l l l l lGTAC ATCACAACTA 30
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CH.9R~CTERISTICS:
(A) LENGTH: 17 Base pairs
~13) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc= "Oligodesoxyribonucleotide"
(iii) HYPOl~TICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GTAAAACGAC GGCCAGT 17
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other nucleic acid
(A) DESCRIPTION: /desc = "Oligodesoxyribonucleotide"
(iii? HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature
43001~NGL.DOC
-
CA 02214430 1997-09-02
44
~B) LOCATION: 1
(D) OTHER INFORMATION:/note= "A at the 5'-terminus is bound via
aminomodifier ~Boehringer l!~nnh~im GmbH) to digoxigenin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
AACAGCTATG ACCATGA 17
43Q~.Nr.r. r~oc
