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Description  |
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FIELD OF THE INVENTION
The present invention relates to methods, systems, and kits-useful for the
identification of molecules that specifically bind to defined nucleic acid
sequences. Also described are methods for designing molecules having the
ability to bind defined nucleic acid sequences and compositions thereof.
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BACKGROUND OF THE INVENTION
Several classes of small molecules that interact with double-stranded DNA
have been identified. Many of these small molecules have profound
biological effects. For example, many aminoacridines and polycyclic
hydrocarbons bind DNA and are mutagenic, teratogenic, or carcinogenic.
Other small molecules that bind DNA include: biological metabolites, some
of which have applications as antibiotics and antitumor agents including
actinomycin D, echinomycin, distamycin, and calicheamicin; planar dyes,
such as ethidium and acridine orange; and molecules that contain heavy
metals, such as cisplatin, a potent antitumor drug.
The sequence binding preferences of most known DNA binding molecules have
not, to date, been identified. However, several small DNA-binding
molecules have been shown to preferentially recognize specific nucleotide
sequences, for example: echinomycin has been shown to preferentially bind
the sequence [(A/T)CGT]/[ACG(A/T)](Gilbert et al.); cisplatin has been
shown to covalently cross-link a platinum molecule between the N7 atoms of
two adjacent deoxyguanosines (Sherman et al.); and calicheamicin has been
shown to preferentially bind and cleave the sequence TCCT/AGGA (Zein et
al.).
Many therapeutic DNA-binding molecules (such as distamycin) that were
initially identified based on their therapeutic activity in a biological
screen have been later determined to bind DNA. There are several examples
in the literature referring to synthetic or naturally-occurring polymers
of DNA-binding drugs. Netropsin, for example, is a naturally-occurring
oligopeptide that-binds to the minor groove of double-stranded DNA.
Netropsin contains two 4-amino-1-methylpyrrole-2-carboxylate residues and
belongs to a family of similar biological metabolites from Streptomyces
spp. This family includes distamycin, anthelvencin (both of which contain
three N-methylpyrrole residues), noformycin, amidomycin (both of which
contain one N-methylpyrrole residue) and kikumycin (which contains two
N-methylpyrrole residues, like netropsin) (Debart, et al.). Synthetic
molecules of this family have also been described, including the
above-mentioned molecules (Lown, et al. 1985) well as dimeric derivatives
(Griffin et al., Gurskii, et al.) and certain analogues (Bialer, et al.
1980, Bialer, et al. 1981, Krowicki, et al.).
Molecules in this family, particularly netropsin and distamycin, have been
of interest because of their biological activity as antibacterial (Thrum
et al., Schuhmann, et al.), antiparasitic (Nakamura et al.), and antiviral
drugs (Becker, et al., Lown, et al. 1986, Werner, et al.).
Among the synthetic analogs of netropsin and distamycin are oligopeptides
that have been designed to have sequence preferences different from their
parent molecules. Such oligopeptides include the "lexitropsin" series of
analogues. The N-methlypyrrole groups of the netropsin series were
systematically replaced with N-methylimidazole residues, resulting in
lexitropsins with increased and altered sequence specificities from the
parent compounds (Kissinger, et al.). Further, a number of
poly(N-methylpyrrolyl)-netropsin analogues have been designed and
synthesized which extend the number of residues in the oligopeptides to
increase the size of the binding site (Dervan, 1986).
There are several different approaches that could be taken to look for
small molecules that specifically inhibit the interaction of a given
DNA-binding protein with its binding sequence (cognate site). One approach
would be to test biological or chemical compounds for their ability to
preferentially block the binding of one specific DNA:protein interaction
but not others. Such an assay would depend on the development of at least
two, preferably three, DNA:protein interaction systems in order to
establish controls for distinguishing between general DNA-binding
molecules (polycations like heparin or intercalating agents like ethidium)
and DNA-binding molecules having sequence binding preferences that would
affect protein/cognate binding site interactions in one system but not the
other(s).
One illustration of how this system could be used is as follows. Each
cognate site could be placed 5' to a reporter gene (such as genes encoding
.beta.-galactoside or luciferase) such that binding of the protein to the
cognate site would enhance transcription of the reporter gene. The
presence of a sequence-specific DNA-binding drug that blocked the
DNA:protein interaction would decrease the enhancement of the reporter
gene expression. Several DNA enhancers could be coupled to reporter genes,
then each construct compared to one another in the presence or absence of
small DNA-binding test molecules. In the case where multiple
protein/cognate binding sites are used for screening, a competitive
inhibitor that blocks one interaction but not the others could be
identified by the lack of transcription of a reporter gene in a
transfected cell line or in an in vitro assay. Only one such DNA-binding
sequence, specific for the protein of interest, could be screened with
each assay system. This approach has a number of limitations including
limited testing capability and the need to construct the appropriate
reporter system for each different protein/cognate site of interest.
Another example of a system to detect sequence-specific DNA-binding
molecules would involve cloning a DNA-binding protein of interest,
expressing the protein in an expression system (e.g., bacterial,
baculovirus, or mammalian expression systems), preparing a purified or
partially purified sample of protein, then using the protein in an in
vitro competition assay to detect molecules that blocked the DNA:protein
interaction. These types of systems are analogous to many receptor:ligand
or enzyme:substrate screening assays developed in the past, but have the
same limitations as outlined above in that a new system must be developed
for every different protein/cognate site combination of interest. The
capacity for screening numerous different sequences is therefore limited.
Another example of a system designed to detect sequence-specific
DNA-binding drugs would be the use of DNA footprinting procedures as
described in the literature. These methods include DNase I or other
nuclease footprinting (Chaires, et al.), hydroxy radical footprinting
(Portugal, et al.), methidiumpropyl EDTA(iron) complex footprinting
(Schultz, et al.), photofootprinting (Jeppesen, et al.), and bidirectional
transcription footprinting (White, et al.). These procedures are likely to
be accurate within the limits of their sequence testing capability but are
seriously limited by (i) the number of different DNA sequences that can be
used in one experiment (typically one test sequence that represents the
binding site of the DNAbinding protein under study), and (ii) the
difficulty of developing high throughput screening systems.
SUMMARY OF THE INVENTION
In one aspect, the invention includes a method of constructing a
DNA-binding agent capable of sequence-specific binding to a duplex DNA
target region. The method includes identifying in the duplex DNA, a target
region containing a series of at least two non-overlapping base-pair
sequences of four base-pairs each, where the four base-pair sequences are
adjacent, and each sequence is characterized by sequence-preferential
binding to a duplex DNA-binding small molecule. The small molecules are
coupled to form a DNA-binding agent capable of sequence-specific binding
to said target region.
In one embodiment, the duplex-binding small molecules are identified as
molecules capable of binding to a selected test sequence in a duplex DNA
by first adding a molecule to be screened to a test system composed of (a)
a DNA-binding protein that is effective to bind to a screening sequence in
a duplex DNA, with a binding affinity that is substantially independent of
the test sequence adjacent the screening sequence, but that is sensitive
to binding of molecules to such test sequence, when the test sequence is
adjacent the screening sequence, and (b) a duplex DNA having said
screening and test sequences adjacent one another, where the binding
protein is present in an amount that saturates the screening sequence in
the duplex DNA.
The test molecule is incubated in the test system for a period sufficient
to permit binding of the molecule being tested to the test sequence in the
duplex DNA. The degree of binding protein bound to the duplex DNA before
adding the test molecule is compared with that after adding the molecule.
The screening sequence may be from the HSV origin of replication, and the
binding protein may be UL9. Exemplary screening sequences are identified
as SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:615, and SEQ ID NO:641.
Specific examples of tetrameric basepair sequences include TTTC, TTTG,
TTAC, TTAG, TTGC, TTGG, TTCC, TTCG, TATC, TATG, TAAC, TAAG, TAGC, TAGG,
TACC, TAGC sequences. A specific example of a small molecule capable of
binding to these sequences is distamycin.
In another aspect, the invention includes a method of blocking
transcriptional activity from a duplex DNA template. The method includes
identifying in the duplex DNA, a binding site for a transcription factor
and, adjacent the binding site, a target region having a series of at
least two non-overlapping tetrameric base-pair sequences, where the four
(tetrameric) basepair sequences are adjacent and each sequence is
characterized by sequence-preferential binding to a duplex DNA-binding
small molecule. The sequences are contacted with a binding agent composed
of the small molecules coupled to form a DNA-binding agent capable of
sequence-specific binding to said target region.
The target may be selected, for example, from DNA sequences adjacent a
binding site for a eucaryotic transcription factor, such as transcription
factor TFIID, or a procaryotic transcription factor, such as transcription
sigma factor.
For mammalian transcription factors, the target region is typically chosen
from non-conserved regions adjacent the transcription factor binding site.
Target regions can be chosen so that the small molecule binding overlaps
an adjacent transcription factor DNA binding sequence (e.g., for a TFIID
binding site, by 1-3 nucleotide pairs). In this case, the specificity of
DNA binding for the small molecule is essentially derived from the
non-conserved sequences adjacent the transcription factor binding site, in
order to reduce small molecule binding at the transcription factor binding
site associated with other genes.
Also disclosed is a DNA-binding agent capable of binding with base-sequence
specificity to a target region in duplex DNA, where the target region
contains at least two adjacent four base-pair sequences. The agent
includes at least two subunits, where each subunit is a small molecule
which has a sequence-preferential binding affinity for a sequence of four
base-pairs in the target region. The subunits are coupled to form a
DNA-binding agent capable of sequence-specific binding to said target
region.
In one general embodiment, the agent is designed for binding to a sequence
in which the two tetrameric basepair sequences are separated (for example,
by up to 20 basepairs, typically, 1 to 6 basepairs) and the small
molecules in the agent are coupled to each other by a spacer molecule.
Also forming part of the invention is a method of constructing a binding
agent capable of sequencespecific binding to a duplex DNA target region.
The method includes identifying in the duplex DNA, a target region
containing (i) a series of at least two adjacent non-overlapping base-pair
sequences of four base-pairs each, where each four base-pair sequence is
characterized by sequence-preferential biding to a duplex DNA-binding
small molecule, and (ii) adjacent to (i) a DNA duplex region capable of
forming a triplex with a third-strand oligonucleotide. The two small
molecules are coupled to form a DNA-binding agent capable of
sequence-specific binding to said target region, and the DNA-binding agent
is attached to a third-strand oligonucleotide.
The binding of the DNA-binding agent to duplex DNA causes a shift from B
form to A form DNA, allowing triplex binding between the third-strand
polynucleotide and a portion of the target sequence.
Also disclosed is a triple-strand forming agent for use in practicing the
method.
In still another aspect, the invention includes a method of ordering the
sequence binding preferences a DNA-binding molecule. The method includes
adding a molecule to be screened to a test system composed of (a) a
DNA-binding protein that is effective to bind to a screening sequence in a
duplex DNA with a binding affinity that is substantially independent of
such test sequence adjacent the screening sequence, but that is sensitive
to binding of molecules to such test sequence, and (b) a duplex DNA having
said screening and test sequences adjacent one another, where the binding
protein is present in an amount that saturates the screening sequence in
the duplex DNA. The molecule in the test system is incubated for a period
sufficient to permit binding of the molecule being tested to the test
sequence in the duplex DNA, and the amount of binding protein bound to the
duplex DNA before and after addition of the test molecule is compared.
These steps are repeated using all test sequences of interest, and the
sequences are then ordered on the basis of relative amounts of protein
bound in the presence of the molecule for each test sequence.
The test sequences are selected, for example, from the group of 256
possible four base sequences composed of A, G, C and T. The DNA screening
sequence is preferably from the HSV origin of replication, and the binding
protein is preferably UL9.
The invention also includes, a method for altering the binding
characteristics of a DNA-binding protein to a duplex DNA. In the method, a
binding site for the DNA-binding protein is identified in the duplex DNA
and a target region identified adjacent the binding site. A small molecule
is selected that is characterized by sequence-preferential binding to the
target region. Such molecules can be selected by the assay and methods of
the present invention. When the small molecule is bound to the target
region, the small molecule is typically adjacent to the binding site for
the DNA-binding protein. Alternatively, the binding of the small molecule
may overlapping the site for the DNA-binding protein by at least one
nucleotide pair. In the case of such overlap, the specificity of DNA
binding for the small molecule is essentially derived from non-conserved
sequences adjacent the DNA-binding protein's binding site--in order to
reduce small molecule binding at similar DNA:protein binding sites at
other locations. Finally, the duplex DNA is contacted with the small
molecule at a concentration effective to alter binding of the DNA-binding
protein to its binding site.
In this method, contacting the duplex DNA with a small molecule can either
inhibit or enhance the binding of the DNA-binding protein to its binding
site: depending on the small molecule that is selected. Exemplary DNA
binding proteins include DNA replication factors and a variety of
transcription factors.
One application of this method is to eucaryotic general transcription
factors (e.g., TFIID), where the target region is typically selected from
DNA sequences adjacent the binding site for the eucaryotic transcription
factor (e.g., SEQ ID NO:1 to SEQ ID NO:600). In one embodiment, the DNA
binding protein is a eucaryotic general transcription factor and the small
molecule binds, in addition to the target region, 1 to three nucleotide
pairs of the DNA-binding protein's binding site. In the case of TFIID, the
small molecule typically binds to (i) the target region, and (ii) up to
two nucleotides of the binding site for TFIID, where the nucleotides are
contiguous to the target region.
Generally, the present invention provides a method of screening for
molecules capable of binding to a selected test sequence in a duplex DNA.
In the method of the present invention a test sequence of interest is
selected. Such sequences can be selected, for example, from the group of
sequences presented as SEQ ID NO:1 to SEQ ID NO:600. Alternatively, the
test sequences can be sequences having randomly generated sequences or
defined sets of sequences, such as, the group of 256 possible four base
sequences composed of A, G, C and T.
A duplex DNA test oligonucleotide is constructed having a screening
sequence adjacent a selected test sequence, where a DNA binding protein is
effective to bind to the screening sequence with a binding affinity that
is substantially independent of the adjacent test sequence. In such
constructs the DNA protein binding to the screening sequence is sensitive
to binding of test molecules to the test sequence.
Molecules selected for testing/screening are added to a test system
composed of (a) the DNA binding protein, and (b) the duplex DNA test
oligonucleotide, which contains the screening and test sequences adjacent
one another. Selected molecules are incubated in the test system for a
period sufficient to permit binding of the molecule being tested to the
test sequence in the duplex DNA. The amount of binding protein bound to
the duplex DNA is compared before and after adding a test molecule.
Comparison of the amount of binding protein bound to the duplex DNA before
and after adding a test molecule can be accomplished, for example, using a
gel band-shift assay or a filter-binding assay.
In the method of the present invention a number of DNA:protein interactions
may be used for screening purposes. In one embodiment, the DNA screening
sequence is from the HSV origin of replication and the binding protein is
UL9. Exemplary HSV origin of replication screening sequences include SEQ
ID NO:601, SEQ ID NO:602, SEQ ID NO:615, and SEQ ID NO:641.
Other DNA:protein interactions useful in the practice of the present
invention include restriction endonucleases and their cognate DNA-binding
sequences. These reactions are typically carried out in the absence of
divalent cations.
In another embodiment, the invention includes a method of identifying test
sequences in duplex DNA to which binding of a test molecule is most
preferred. In this method a mixture of duplex DNA test oligonucleotides is
constructed, where each oligonucleotide has a screening sequence adjacent
a test sequence as described above. The test oligonucleotides of the
mixture typically contain different test sequences.
A test molecule, to be screened, is added to a test reaction composed of
(a) the DNA binding protein, and (b) the duplex DNA test oligonucleotide
mixture. The molecule is incubated in the test reaction for a period
sufficient to permit binding of the compound being tested to test
sequences in the duplex DNA. Test oligonucleotides are separated from test
oligonucleotides bound to binding protein.
The test oligonucleotides can be separated from test oligonucleotides bound
to protein by, for example, passing the test reaction through a filter,
where the filter is capable of capturing DNA:protein complexes but not DNA
that is free of protein. One filter type useful in the practice of the
present invention is the nitrocellulose filter.
The separated test oligonucleotides are then amplified. These amplified
test oligonucleotides are then recycled through the screening steps of the
assay in order to obtain a desired degree of selection. The amplified test
oligonucleotides are isolated and sequenced.
Exemplary test sequences include sequences selected from the group of 256
possible four base sequences composed of A, G, C and T. Further examples
of desirable test sequences include test sequences derived from the
sequences presented as SEQ ID NO:1 to SEQ ID NO:600.
The amplification step in the method may be accomplished by polymerase
chain reaction or other methods of amplification, including, cloning and
subsequent in vivo amplification of the cloning vector containing the
sequences of interest.
These and other objects and features of the invention will be more fully
appreciated when the following detailed description of the invention is
read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A illustrates a DNA-binding protein binding to a screening sequence.
FIGS. 1B and 1C illustrate how a DNA-binding protein may be displaced or
hindered in binding by a small molecule by two different mechanisms:
because of stearic hinderance (1B) or because of conformational
(allosteric) changes induced in the DNA by a small molecule (1C).
FIG. 2 illustrates an assay for detecting inhibitory molecules based on
their ability to preferentially hinder the binding of a DNA-binding
protein to its binding site. Protein (O) is displaced from DNA (/) in the
presence of inhibitor (X). Two alternative capture/detection systems are
illustrated, the capture and detection of unbound DNA or the capture and
detection of DNA:protein complexes.
FIG. 3 shows a DNA-binding protein that is able to protect a biotin moiety,
covalently attached to an oligonucleotide sequence, from being recognized
by streptavidin when a protein is bound to the DNA.
FIG. 4 shows the incorporation of biotin and digoxigenin into a typical
oligonucleotide molecule for use in the assay of the present invention.
The oligonucleotide contains the binding sequence (i.e., the screening
sequence) of the UL9 protein, which is underlined, and test sequences
flanking the screening sequence. FIG. 4 also shows the preparation of
double-stranded oligonucleotides end-labeled with either digoxigenin or
.sup.32 p.
FIG. 5 shows a series of sequences that have been tested in the assay of
the present invention for the binding of sequence-specific small
molecules.
FIG. 6 outlines the clonings, into an expression vector, of a truncated
form of the UL9 protein (UL9COOH) which retains its sequence-specific
DNA-binding ability.
FIG. 7 shows the pVL1393 baculovirus vector containing the full length UL9
protein coding sequence.
FIG. 8 is a photograph of a SDS-polyacrylamide gel showing (i) the purified
UL9-COOH/glutathione-Stransferase fusion protein and (ii) the UL9-COOH
polypeptide.
FIG. 9 presents data demonstrating the effect on UL9-COOH binding of
alterations in the test sequences that flank the UL9 screening sequence.
FIG. 10A shows the effect of the addition of several concentrations of
distamycin A to DNA:protein assay reactions utilizing different test
sequences. FIG. 10B shows the effect of the addition of actino-mycin D to
DNA:protein assay reactions utilizing different test sequences. FIG. 10C
shows the effect of the addition of Doxorubicin to DNA:protein assay
reactions utilizing different test sequences.
FIG. 11A illustrates a DNA capture system of the present invention
utilizing biotin and streptavidin coated magnetic beads. The presence of
the DNA is detected using an alkaline-phosphatase substrate that yields a
chemiluminescent product. FIG. 11b shows a similar reaction using biotin
coated agarose beads that are conjugated to streptavidin, that in turn is
conjugated to the captured DNA.
FIG. 12 demonstrates a test matrix based on DNA:protein-binding data.
FIG. 13 lists the top strands (5'-3') of all the possible four base pair
sequences that could be used as a defined set of ordered test sequences in
the assay.
FIG. 14A lists the top strands (5'-3') of all the possible four base pair
sequences that have the same base composition as the sequence 5'-GATC-3'.
This is another example of a defined, ordered set of sequences that could
be tested in the assay. FIG. 14B presents the general sequence of a test
oligonucleotide (SEQ ID NO:617); where XXXX is the test sequence and
N=A,G,C, or T.
FIG. 15 shows the results of 4 duplicate experiments in which the binding
activity of distamycin was tested with all possible (256) four base pair
sequences. The oligonucleotides are ranked from 1 to 256 (column 1,
"rank") based on their average rank from the four experiments (column 13,
"ave. rank"). (rank is shown in the first column of the chart).
FIG. 16 shows the average ranks (FIG. 15) plotted against the ideal ranks 1
to 256.
FIG. 17 shows the average r% scores (FIG. 15) plotted against the rank of 1
to 256.
FIG. 18 shows the results of eight experiments with actinomycin D. The r%
scores and rank are shown for each of the 256 oligonucleotides.
FIG. 19 shows the average r% versus rank, by average rank (data from FIG.
18).
FIG. 20 shows the ideal and average ranks for each of the 256
oligonucleotides.
FIG. 21 shows the results of a position analysis for actinomycin D
preference.
FIG. 22 presents the data for a dinucleotide analysis of actinomycin D
binding preference.
FIG. 23 graphically displays the results presented in FIG. 22.
FIG. 24 graphically displays the data presented in FIG. 22, where the data
are combined in a combined bar chart so that the cumulative results for
any dinucleotide pair are tabulated in a single bar.
FIG. 25 shows the top strands of 16 possible duplex DNA target sites for
binding bis-distamycins.
FIG. 26 shows examples of bis-distamycin target sequences for
bis-distamycins with internal flexible and/or variable length linkers
targeted to sites comprised of two TTCC sequences, where N is any base.
FIGS. 27A to 27H show sample oligonucleotides for competition binding
studies using the assay of the present invention.
FIG. 28 shows the DNA sequences of the HIV proviral promoter region.
Several transcription factor binding sites are marked.
FIGS. 29A to 29D illustrate sample test oligonucleotides fer use in the
polymerase chain reaction based selection technique of the present
invention. In FIG. 29A, X is the number of bases that comprise the test
site.
FIG. 30 illustrates a sample test oligonucleotide for use in the assay of
the present invention, where the test oligonucleotide employs several
different DNA:protein interaction systems.
FIG. 31 illustrates the results of screening a selected test sequence with
a single DNA:protein interaction system. In the figure, the test site is
shown in bold, the potential binding site for the test molecule is
underlined.
FIG. 32 illustrates the results of screening the same selected test
sequence as shown in FIG. 31, but using a different single DNA:protein
interaction system. In the figure, the test site is shown in bold, the
potential binding site for the test molecule is underlined.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
Adjacent is used to describe the distance relationship between two
neighboring sites. Adjacent sites are 20 or less bp apart, and can be
separated by any fewer number of bases including the situation where the
sites are immediately abutting one another. "Flanking" is a synonym for
adjacent.
Bound DNA, as used in this disclosure, refers to the DNA that is bound by
the protein used in the assay (e.g., a test oligonucleotide containing the
UL9 binding sequence bound to the UL9 protein.
Coding sequences or coding regions are DNA sequences that code for RNA
transcripts, unless specified otherwise.
Dissociation is the process by which two molecules cease to interact: the
process occurs at a fixed average rate under specific physical conditions.
Functional binding is the noncovalent association of a protein or small
molecule to the DNA molecule. In one embodiment of the assay of the
present invention the functional binding of the UL9 protein to a screening
sequence (i.e., its cognate DNA binding site) has been evaluated using
filter binding or gel band-shift experiments.
Half-life is herein defined as the time required for one-half of the
associated complexes, e.g., DNA:protein complexes, to dissociate.
Heteropolymers are molecules comprised of at least two different subunits,
each representing a different type or class of molecule. The covalen | | |
