WO1999032664A1

WO1999032664A1 - Method of selecting flanking sequences which convey relative binding affinities to a ligand binding site

Info

Publication number: WO1999032664A1
Application number: PCT/US1998/027461
Authority: WO
Inventors: Michael J. Lane; Albert S. Benight; Brian D. Faldasz
Original assignee: Tm Technologies, Inc.
Priority date: 1997-12-23
Filing date: 1998-12-23
Publication date: 1999-07-01
Also published as: AU2011499A; EP1042510A1; JP2001526905A

Abstract

Methods of ranking the relative reactivity of nucleotide sequences flanking a ligand binding site. The invention involves, and is useful for, ranking populations of nucleic acid molecules, each containing a ligand binding site and flanking sequences on either side of the binding site. In an embodiment a method of ranking reactivities of polynucleotide flanking sequences is disclosed wherein the method includes (a) providing a plurality of different nucleic acid molecules, wherein each of the molecules has the same ligand binding site located adjacent to at least one flanking sequence; (b) exposing the molecules to a ligand selective for the binding site under conditions such that the relative binding affinity of the ligand for the binding site within at least two of the molecules is determined; and (c) ranking the relative binding affinities determined in step (b) to rank the relative reactivities of said polynucleotide flanking sequences. In another embodiment, a higher relative binding affinity for the ligand binding site within the molecule(s) corresponds to a higher relative reactivity of the flanking sequence(s).

Description

METHOD OF SELECTING FLANKING SEQUENCES WHICH CONVEY RELATIVE BINDING AFFINITIES TO A LIGAND BINDING SITE

Field of the Invention This invention features a method of ranking relative reactivities of nucleotide sequences and related methods and products. In accordance with this invention, sequences can be graded into a range of reactivity from highly reactive to less reactive.

Background of the Invention DNA is known to undergo a wide variety of conformational alterations which are dependent on the conditions in which the DNA is found. Critical to the final structure(s) adopted by DNA are the precise order of bases, the length of the DNA, the overall base content (GC/AT content) and the stability of the sequence to thermal denaturation. While not widely appreciated, some sequences of DNA can convert relatively easily between different conformational states. An example of this type of behavior is the well documented conversion of poly dGC to a Z (left-handed helix) as opposed to the normal right-handed B form in the presence of a high salt environment (Pohl, FM and Jovin, TM (1972) J. Mo!. Biol. 67, 375-396). While this conformational flexibility has been exhibited in many other simple repeated sequences, in the vast majority of such cases the final "altered" conformation has not been characterized to the extent of the Z DNA example described above. This suggests that such conformational variability is a common albeit unappreciated feature of DNA. Interestingly, such sequences are also known to be highly reactive to common endonucleases. Underscoring this point are studies which indicate that small (GC)g DNA segments can influence the overall structure of apparently random sequence DNA segments as large as one thousand bases in length (Kirn et al., (1993) Biopolymers 33, 1725-1745). In toto the current understanding of this phenomenon is insufficient to permit useful information to be obtained by simple interpretation of a nucleotide sequence.

Summary of the Invention

The present disclosure details a method of ranking the relative reactivity of nucleotide sequences flanking a ligand binding site. The invention involves, and is useful for, ranking populations of nucleic acid molecules, each containing a ligand binding site and flanking sequences on either side of the binding site. In an embodiment a method of ranking reactivities of polynucleotide flanking sequences is disclosed wherein the method includes (a) providing a plurality of different nucleic acid molecules, wherein each of the molecules has the same ligand binding site located adjacent to at least one flanking sequence; (b) exposing the molecules to a ligand selective for the binding site under conditions such that the relative binding affinity of the ligand for the binding site within at least two of the molecules is determined; and (c) ranking the relative binding affinities determined in step (b) to rank the relative reactivities of said polynucleotide flanking sequences. In a further embodiment, a higher relative binding affinity for the ligand binding site within the molecule(s) corresponds to a higher relative reactivity of the flanking sequence(s).

In an advantageous embodiment, the restriction endonuclease BamHI (DNA binding ligand) may be added to a population of duplex polynucleotide molecules which comprise a BamHI binding site (5'-GGATCC-3') flanked on either side by a sequence of nucleotides under conditions such that the binding affinity of the ligand for the binding site may be determined, i.e., conditions which allow BamHI to contact the binding site but not cleave it. Only a fraction of the duplex polynucleotide molecules will bind to BamHI. The bound duplex polynucleotide molecules can be separated from the unbound duplex polynucleotide molecules by conventional methods, and the nucleotide sequences flanking the BamHI binding site of the bound and unbound duplex polynucleotide molecules determined by known sequencing techniques. The flanking nucleotide sequences are usually randomly synthesized nucleotides, but can also include any predetermined combination of A, T, C or G. Another embodiment relates to methods for isolating and ranking duplex polynucleotide sequences which confer relatively high or low reactivity towards a particular duplex polynucleotide ligand. In these methods, duplex polynucleotide molecules comprise a predetermined ligand binding site which is flanked by a randomly synthesized or other duplex polynucleotide sequence. The duplex polynucleotide molecules are exposed to a ligand selective for the binding site under conditions which promote binding of ligand to the binding site; and ligand-bound duplex polynucleotide molecules are isolated from ligand-unbound duplex polynucleotide molecules. In a preferred embodiment, the duplex polynucleotide molecules are sequenced to determine the sequence identity of the duplex polynucleotide sequence flanking the ligand binding site.

In another aspect, a method is disclosed for isolating duplex polynucleotide molecules with relatively high or relatively low binding affinity to a given ligand from a plurality of duplex polynucleotide molecules, comprising (a) providing a plurality of different duplex polynucleotides, wherein each of the molecules has the same ligand binding site, and a randomly synthesized polynucleotide sequence flanking the binding site; (b) exposing the different duplex polynucleotide molecules to a ligand selective for the binding site under conditions which promote the binding of ligand to the binding site; and (c) isolating duplex polynucleotide molecules which bind to the ligand. The isolated duplex polynucleotide molecules may advantageously further be amplified and sequenced to determine the sequence identity of the duplex polynucleotide sequence flanking the ligand binding site. The invention further provides a method of ranking the flanking duplex polynucleotide sequences based on their ability to influence the binding of the ligand to its binding site.

Brief Description of the Drawings

FIG. 1 shows a schematic representation of the selection of subpopulations of polynucleotide molecules having higher or lower relative reactivity for the restriction endonuclease BamHI.

FIG. 2 shows an ethidium bromide-stained native polyacrylamide gel of the products of an amplification reaction using the polymerase chain reaction, wherein some of the products have undergone restriction endonuclease digestion.

FIG. 3 shows an ethidium bromide-stained native polyacrylamide gel of a BamHI band shift assay.

Detailed Description of the Invention

The present disclosure will be more fully illustrated by reference to the definitions set forth below.

"Polynucleotide" is intended to include multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). The term "polynucleotide" as used herein, refers to polyribonucleotides and polydeoxyribonucleotides. Polynucleotides can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but may also be synthetic DNA or RNA (e.g., produced by oligonucleotide synthesis).

"Ligand" is intended to include any chemical moiety that can interact covalently or non-covalently with either a single strand or duplex nucleic acid molecule. Non- limiting examples of ligands include compounds which bind to a polynucleotide sequence in a sequence-specific or non-specific way; proteins; enzymes, e.g., restriction enzymes; restriction endonucleases; ligases such as DNA ligase; nucleic acid polymerases such as Taq polymerase; topoisomerases; DNA binding agents; mutagens; compounds which enhance the expression of a gene under the control of the duplex polynucleotide sequence bound by a ligand; compounds which intercalate into a duplex polynucleotide molecule; compounds which, when contacted with a reaction mixture comprising a first single stranded polynucleotide molecule and a second single stranded polynucleotide molecule will increase the free energy of duplex formation at least n-fold, wherein n is 2, 5, 10, 50 100, 500, 10³, 10⁴, 10⁵, 10⁶; and compounds which, when contacted with a reaction mixture will decrease the free energy of duplex formation by at least n-fold, wherein n is 2, 5, 10, 50, 100, 500, 10³, 10⁴, 10⁵, 10⁶.

"Ligand binding site" or "binding site" is intended to include any domain or subdomain in a nucleic acid molecule which directly contacts a ligand by hydrogen bonding, van der Waals radius interactions, electron cloud interaction with the bases of a nucleic acid molecule or indirectly via a salt or water molecule.

"Flanking sequence" is intended to include a polynucleotide sequence located adjacent a ligand binding site of a nucleic acid molecule.

"Relative binding affinities" of flanking sequences are measured for different nucleic acid molecules in which different flanking sequences are located adjacent the same ligand binding site. Where a first molecule having a first flanking sequence (or pair of flanking sequences) is found to preferentially bind to a ligand compared to a second molecule having a second flanking sequence (or pair of flanking sequences), the first molecule is said to have a binding affinity that is "relatively higher" than the binding affinity of the second molecule.

"Relative reactivity" of a flanking sequence (or a pair of flanking sequences) correlates with the relative binding affinity of a molecule containing the sequence(s). A flanking sequence (or pair of flanking sequences) which confers a relatively high binding affinity on a binding site is thus said to have a relatively high reactivity. An endonuclease is said to be "substantially free of cleavage activity" when under the given conditions, there is substantially no observable cleavage.

The present invention may be further illustrated by the following non-limiting examples describing the isolation and characterization of duplex polynucleotide flanking sequences which affect relative reactivities of a binding site flanked by these sequences.

EXAMPLE 1

Figure 1 illustrates a design of a selection scheme for selection of subpopulations from a random population of sequences flanking a BamHI binding site, e.g., flanking which confer a specified relative reactivity to a polynucleotide for BamHI. The example is not intended to be limiting for BamHI; this method may be used with any ligand. Shown in the box at the top is a linear DNA sequence construct created by synthetic means known in the art and consists of (from left to right, 5'-3') a unique PCR primer site followed by a random insert of 20, 40 or 80 bases created by allowing a DNA synthesizer to insert any of the four nucleotide bases A, G, C, and T; a BamHI binding site; and a second 20, 40 or 80 base random insert followed by a second unique PCR primer site. Each primer site includes an appropriate restriction site, e.g., for purposes of cloning said selected sequences for preparation of libraries and sequence determination. When synthesized, a population of synthetic polynucleotide molecules are generated with different polynucleotide sequences at the random insert sites which, after PCR amplification using oligonucleotides complementary to the primer sites as PCR primers, are transformed into a population of duplex polynucleotide molecules containing a BamHI recognition site flanked by random polynucleotide sequences.

Incubation of these duplexes with appropriate (empirically determined) quantities of the endonuclease results in a portion of the duplexes being bound by BamHI while some of the duplexes are not bound. These sequences which bind BamHI with a relatively high affinity bind the endonuclease in preference to those sequences which bind BamHI with a relatively low affinity. Since the bound duplexes can be separated from the unbound duplexes in a gel-shift assay, those duplexes bound to the enzyme with higher affinity are represented as "shifted" duplexes at relatively lower BamHI concentrations. This assay is depicted in Figure 1 as an inset. Lane 1 shows schematically the migration pattern of the unbound duplex population. Lane 2 represents the migration pattern obtained at relatively low BamHI concentrations. Lanes 3 and 4 show how the migration pattern varies as the concentration of BamHI is increased still further.

The population of bound and unbound sequences are eluted separately from the gel and libraries are constructed by digesting at the unique PCR primer sites and subsequently cloned into a vector, propagated and isolated. Sequencing of these clones reveals sequence motifs that confer higher or lower affinity for the endonuclease.

EXAMPLE 2

This example describes the formation of duplex polynucleotide molecules from randomly synthesized single stranded polynucleotide molecules with higher or lower relative affinities for a ligand.

Duplex polynucleotide molecules containing 40 nucleotide inserts adjacent to the BamHI site, are generated by the polymerase chain reaction (PCR) in 100 μl reactions containing one (1) pmole of the single stranded polynucleotide molecules generated synthetically, 200 pmoles of each of the 5' and 3' primers, 1.5 mM MgCl, 0.4 mM each dATP; dGTP; dCTP; and dTTP, and 5U of Taq DNA polymerase in 10 mM Tris-HCl (pH 9.0 at 25°C), to mM KC1, and 0.1% Triton X-100. First round PCR conditions are 1 min at 94°C; 1 min at 48°C; and 10 min at 74°C; followed by 50 cycles of 1 min at 92° C; 1 min at 48°C; and 1 min at 74°C. The products from several such reactions are combined, concentrated to 50 μl in 50 mM Tris-HCl pH 8.0, 50 mM NaCl using a Centricon 30 concentrator (Amicon) and subsequently separated by electrophoresis on a 8% native polyacrylamide gel preequilibrated at 20°C. The separated polynucleotide molecules are visualized by UV light (backshadowed) and molecules of the desired size excised and eluted out of the gel slices in 1ml of 50 mM Tris-HCl pH 8.0, 50 mM NaCl and further concentrated in 50 μl of the same buffer. The concentration of the purified and duplex polynucleotide is then measured by spectrophotometer at 260nm. To confirm that the ligand binding site synthesized is correct and that the PCR did not alter the BamHI binding site, aliquots of the products of the PCR are subjected to restriction digest analysis using 20U of either BamHI or EcoRI as a control. The products of the reaction are then separated by electrophoresis on the same gel next to the undigested control reaction. Results are shown in Figure 2. Lanes 1 and 7 show molecular weight markers. Lanes 2 and 3 show the undigested products of the PCR carried out as described above after the bands of the desired size have been excised. Lane 4 shows 100 ng of the undigested products of the PCR carried out as described above. Lane 5 shows 100 ng of the PCR product digested with the restriction endonuclease EcoRI. Lane 6 shows 100 ng of the PCR product digested with the restriction endonuclease BamHI. As EcoRI should not be able to recognize the BamHI site, the mobility of the undigested duplex polynucleotide molecules will be similar to the mobility of duplex polynucleotide molecules digested with EcoRI. A comparison of lanes 4 and 5 shows that this is indeed the case. If the BamHI site is correctly synthesized and the PCR does not introduce any mutations within the BamHI site, BamHI will bind to its site and cleave the duplex polynucleotide molecule in half. Lane 6 shows that PCR product digested with BamHI runs at a molecular weight approximately half the size of the undigested or EcoRI digested PCR product.

EXAMPLE 3

This example illustrates a method of isolating populations of duplex polynucleotide molecules with higher or lower relative affinities for a ligand. Subsequently, one can determine the sequences of the randomly synthesized duplex molecules which confer that relative binding affinity. To identify populations of duplex polynucleotides with relatively high or low affinity to BamHI, a band shift assay is performed. 100 ng aliquots of the PCR amplified duplex polynucleotide molecules containing 0.1 pmol of 32p end labeled duplex polynucleotide is incubated with variable concentrations of BamHI in a total volume of 30 μl of 50 mM Tris-HCl pH 8; 50 mM EDTA; 50 mM NaCl; 1 mM DTT for 1 hr at 37°C. Under these conditions BamHI will contact its binding site within the duplex polynucleotide molecule but not cleave it. The "shifted" and "unshifted" bands are separated by electrophoresis on an 8% native polyacrylamide gel. The "shifted" and "unshifted" bands are further excised and eluted out of the gel slice overnight in 1 ml of 50 mM Tris-HCl pH 8.0, 50 mM NaCl. The sample is next concentrated down to 50 μl of the same buffer and amplified by PCR to facilitate further manipulation of the duplex polynucleotide molecules. Results are shown in Figure 3.

Lanes 2 to 10 show the result of incubating a constant amount of duplex polynucleotide with decreasing amounts of BamHI. "Shifted" duplex polynucleotide molecules can be seen in reactions containing as little as 12.5U of BamHI (lane 6). The ratio of "shifted" to "unshifted" duplex polynucleotide molecules increases with increasing amounts of BamHI. Those sequences which bind BamHI with higher affinity are represented as "shifted" duplexes at relatively lower BamHI concentrations.

EXAMPLE 4

This example describes the cloning and subsequent sequencing of the relatively lower and higher reactive duplex polynucleotide molecules.

The "shifted" and "unshifted" duplex polynucleotide molecules amplified in Example 3 are further digested with 100U of the restriction endonucleases EcoRI and Xhol per μg of duplex polynucleotide, the primers in this case including EcoRI sites.

The cleaved duplex polynucleotide molecules are separated by electrophoresis on a native polyacrylamide gel, excised and eluted overnight in 1 ml of 50 mM Tris-HCl pH 8.0, 50 mM NaCl and further concentrated down to 50 μl in the same buffer. 2.8 μg of duplex polynucleotide molecules are ligated into Lambda ZAP II vector predigested with EcoRI and Xhol and treated with CIAP at 1 :1 insert to vector ratio in the presence of 2U of T4 ligase in 5μl of T4 ligase buffer at 40°C overnight. The ligated samples are packaged using Gigapack II Gold packaging extract

(Stratagene) and cloned into E. Coli XL 1 -Blue host strain and subjected to blue/white selection. The recombinant (white) clones are selected and eluted in 500 μl SM buffer (100 mM NaCl, 8mM MgSO 50 mM Tris-HCl pH 7.5, 0.01% gelatin, 0.04% chloroform). 10 μl of the eluate is amplified by PCR using ml3 forward and reverse primers, purified by Qiagen PCR purification kit and sequenced.

It is possible to carry out equivalent procedures without the use of PCR. For example, the molecule of Example 1 can be synthesized, a fill-in reaction (RTase/polymerase) primed with the right side primer, followed by isolation of the resultant duplexes on a gel. This population can then be used for gel shifting and subsequent cloning.

The finding that a particular DNA binding ligand can bind to its binding site differentially in the contact of the randomly synthesized flanking sequences permits the identification of polynucleotide sequences which have the ability to influence the binding affinity of the DNA binding ligand to its binding site and ultimately affect the functioning of the ligand. For example, the method described above could be used to identify polynucleotide sequences flanking the binding site in DNA of a transcription factor to its binding site. In particular, if the method described above for BamHI was carried out utilizing a particular transcription factor such as MyoD (MyoD is a transcription factor which plays a role in muscle development) as the ligand and the sequence 5'-CANNTG-3' where N is any one of the nucleotides A, C, T or G as the ligand binding site between two randomly synthesized duplex polynucleotide sequences, it may be possible to identify polynucleotide sequences flanking the MyoD binding site in the genome which will either enhance binding of the MyoD to its binding site or result in poor binding of MyoD to its binding site. Thus, duplex polynucleotide molecules comprising randomly synthesized polynucleotide sequences flanking either site of the MyoD binding site which can be isolated as bound complexes with MyoD can be said to confer high binding affinity of MyoD to its binding site and duplex polynucleotide sequences which are isolated unbound with MyoD can be said to confer low binding affinity of MyoD to its binding site. Further, the randomly synthesized polynucleotide sequences can be ranked in order of the ability to influence binding affinity of the ligand to its binding site.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference.

Claims

What is claimed is;

1. A method of ranking reactivities of polynucleotide flanking sequences, the method comprising: (a) providing a plurality of different nucleic acid molecules, wherein each of said molecules has the same ligand binding site located adjacent to at least one flanking sequence;

(b) exposing said plurality of molecules to a ligand selective for the binding site, under conditions such that the relative binding affinity of the ligand for the binding site within at least two of said molecules of said plurality of molecules is determined; and

(c) ranking the relative binding affinities determined in step (b) to rank the relative reactivities of said polynucleotide flanking sequences.

2. The method of claim 1 wherein higher relative binding affinity for said ligand binding site within the molecule corresponds to a higher relative reactivity of said flanking sequence.

3. The method of claim 1 wherein said ligand is a restriction endonuclease and the molecules are exposed thereto under conditions in which the endonuclease is substantially free of cleavage activity.

4. The method of claim 3 wherein said molecules are exposed to said restriction endonuclease in a medium sufficiently free of Mg2⁺ to render said endonuclease substantially free of cleavage activity.

5. The method of claim 4, wherein said restriction endonuclease is BamHI.

6. The method of claim 1, wherein said ligand binding site is specific for the ligand selected.

7. The method of claim 1, wherein said flanking sequence is about 20 to 80 nucleotides long.

8. The method of claim 1, wherein said flanking sequence is randomly synthesized.

9. The method of claim 1, wherein said ligand binding site is located between a pair of flanking sequences.

10. The method of claim 1, wherein said ligand binding site is located 5' of said flanking sequence.

11. The method of claim 1 , wherein said ligand binding site is located 3' of said flanking sequence.

12. The method of claim 9, wherein each of said molecules includes a primer sequence for use in amplification of said molecule.

13. The method of claim 12 wherein each molecule includes two of said primers and a first said flanking sequence is located between a first of the primers and the binding site and a second said flanking sequence is located between a second of said primers and the binding site.

14. The method of claim 1 wherein said molecules are amplified prior to step (b).

15. The method of claim 13, further comprising the step of amplifying molecules having a higher relative binding affinity.

16. The method of claim 13, further comprising the step of amplifying molecules having a lower relative binding affinity.

17. The method of claim 15, further comprising the step of sequencing flanking sequences of the molecules having a higher relative binding affinity after amplification of said molecules.

18. The method of claim 16, further comprising the step of sequencing flanking sequences of the molecules having a lower relative binding affinity after amplification of said molecules.

19. The method of claim 1, wherein the step of ranking the binding affinities includes affinity purification or band shifting of said molecules.

20. The method of claim 1 , wherein only a portion of the molecules become bound to the ligand during step (b), and step (c) further comprises separating bound and unbound molecules from each other.

21. A method of isolating duplex polynucleotide molecules with relatively high or relatively low binding affinity to a given ligand from a plurality of duplex polynucleotide molecules, comprising:

(a) providing a plurality of different duplex polynucleotides, wherein each of said duplex polynucleotide molecules has the same ligand binding site, and a randomly synthesized polynucleotide sequence flanking said binding site;

(b) exposing said plurality of different duplex polynucleotide molecules to a ligand selective for the binding site under conditions which promote the binding of ligand to the binding site; (c) isolating duplex polynucleotide molecules which bind to the ligand.

22. The method of claim 21, wherein the ligand is a restriction endonuclease and the molecules are exposed thereto under conditions in which the endonuclease is substantially free of enzymatic activity.

23. The method of claim 22, wherein the molecules are exposed to the restriction endonuclease in a condition sufficiently free of Mg***-⁺ to render the endonuclease substantially free of enzymatic activity.

24. The method of claim 21 , wherein the predetermined ligand binding site is specific for the ligand selected from a group of nucleic acid binding compounds.

25. The method of claim 21 , wherein the randomly synthesized duplex polynucleotide flanking sequences comprise from about 20 to 80 nucleotides.

26. The method of claim 21 , wherein the ligand binding site is located between a pair of flanking sequences.

27. The method of claim 21, wherein the ligand binding site is located 5' of a flanking sequence.

28. The method of claim 21, wherein the ligand binding site is located 3' of a flanking sequence.

29. The method of claim 21, wherein the step of isolating the duplex polynucleotide molecules which bind to the ligand includes affinity purification or band shifting in order to separate duplex polynucleotide molecules bound to a said ligand from unbound duplex polynucleotide molecules.

30. An isolated polynucleotide molecule obtained according to the method of claim 1.

31. An isolated polynucleotide molecule of claim 30 wherein the molecule includes a pair of flanking sequences and first and second primers at first and second ends of the molecules, respectively.