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WO1999032664A1 - Procede de selection de sequences adjacentes porteuses d'affinites de liaison relatives a un site de liaison aux ligands - Google Patents

Procede de selection de sequences adjacentes porteuses d'affinites de liaison relatives a un site de liaison aux ligands Download PDF

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Publication number
WO1999032664A1
WO1999032664A1 PCT/US1998/027461 US9827461W WO9932664A1 WO 1999032664 A1 WO1999032664 A1 WO 1999032664A1 US 9827461 W US9827461 W US 9827461W WO 9932664 A1 WO9932664 A1 WO 9932664A1
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WIPO (PCT)
Prior art keywords
molecules
ligand
binding site
flanking
polynucleotide
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Application number
PCT/US1998/027461
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English (en)
Inventor
Michael J. Lane
Albert S. Benight
Brian D. Faldasz
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Tm Technologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Tm Technologies, Inc. filed Critical Tm Technologies, Inc.
Priority to JP2000525581A priority Critical patent/JP2001526905A/ja
Priority to EP98964895A priority patent/EP1042510A1/fr
Priority to AU20114/99A priority patent/AU2011499A/en
Publication of WO1999032664A1 publication Critical patent/WO1999032664A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • This invention features a method of ranking relative reactivities of nucleotide sequences and related methods and products.
  • sequences can be graded into a range of reactivity from highly reactive to less reactive.
  • 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).
  • 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.
  • a method of ranking reactivities of polynucleotide flanking sequences 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.
  • 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).
  • 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.
  • 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.
  • the duplex polynucleotide molecules are sequenced to determine the sequence identity of the duplex polynucleotide sequence flanking the ligand binding site.
  • a method 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.
  • 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.
  • 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)).
  • cytosine (C), thymidine (T) or uracil (U) a substituted purine
  • A adenine
  • G guanine
  • polynucleotide 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.
  • 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.
  • 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
  • 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.
  • Frlanking sequence is intended to include a polynucleotide sequence located adjacent a ligand binding site of a nucleic acid molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • duplexes 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.
  • 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.
  • 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 ⁇ l 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.
  • PCR polymerase chain reaction
  • 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 ⁇ l 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 ⁇ l of the same buffer.
  • the concentration of the purified and duplex polynucleotide is then measured by spectrophotometer at 260nm.
  • aliquots of the products of the PCR are subjected to restriction digest analysis using 20U of either BamHI or EcoRI as a control.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g of duplex polynucleotide, the primers in this case including EcoRI sites.
  • 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 ⁇ l in the same buffer.
  • 2.8 ⁇ g 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 5 ⁇ l of T4 ligase buffer at 40°C overnight.
  • the ligated samples are packaged using Gigapack II Gold packaging extract
  • Example 1 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.
  • RTase/polymerase a fill-in reaction primed with the right side primer
  • 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.
  • 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.
  • MyoD is a transcription factor which plays a role in muscle development
  • N is any one of the nucleotides A, C, T or G as the ligand binding site between two randomly synthesized duplex polynucleotide sequences
  • 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.
  • the randomly synthesized polynucleotide sequences can be ranked in order of the ability to influence binding affinity of the ligand to its binding site.

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Abstract

L'invention concerne des procédés permettant de classer la réactivité relative de séquences nucléotidiques adjacentes d'un site de liaison aux ligands. Cette invention consiste notamment à classer des populations de molécules d'acide nucléique, chacune de ces molécules contenant un site de liaison aux ligands et des séquences adjacentes, de n'importe quel coté dudit site de liaison. Dans un mode de réalisation, un procédé de cette invention permet de classer les réactivités de séquences polynucléotidiques adjacentes, ce procédé consistant: a) à prendre plusieurs molécules d'acide nucléique différentes, chacune de ces molécules présentant le même site de liaison aux ligands placé à proximité d'au moins une séquence adjacente; b) à exposer ces molécules à un ligand sélectif vis-à-vis dudit site de liaison, dans des conditions permettant de déterminer l'affinité de liaison relative entre ce ligand et ce site de liaison, à l'intérieur d'au moins deux molécules; et c) à classer les affinités de liaison relatives, déterminées au cours de l'étape précédente, afin de classer également les réactivités relatives desdites séquences nucléotidiques adjacentes. Dans un autre mode de réalisation, une affinité de liaison relative supérieure en faveur du site de liaison aux ligands à l'intérieur de la(des) molécule(s) correspond à une réactivité relative supérieure de la(des) séquence(s) adjacente(s).
PCT/US1998/027461 1997-12-23 1998-12-23 Procede de selection de sequences adjacentes porteuses d'affinites de liaison relatives a un site de liaison aux ligands WO1999032664A1 (fr)

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JP2000525581A JP2001526905A (ja) 1997-12-23 1998-12-23 相対的結合親和性をリガンド結合部位にもたらすフランキング配列を選択する方法
EP98964895A EP1042510A1 (fr) 1997-12-23 1998-12-23 Procede de selection de sequences adjacentes porteuses d'affinites de liaison relatives a un site de liaison aux ligands
AU20114/99A AU2011499A (en) 1997-12-23 1998-12-23 Method of selecting flanking sequences which convey relative binding affinities to a ligand binding site

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US6861697P 1997-12-23 1997-12-23
US60/068,616 1997-12-23

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063117A1 (fr) * 1998-06-04 1999-12-09 Tm Technologies, Inc. Procede de creation de sequences polynucleotidiques flanquantes qui vehiculent des affinites de liaison relatives a un site de liaison de ligand
WO1999042621A3 (fr) * 1998-02-21 2000-03-16 Tm Technologies Inc Proprietes thermodynamiques d'acides nucleiques
WO1999063074A3 (fr) * 1998-06-04 2000-04-06 Tm Technologies Inc Methode permettant de modifier le niveau d'expression relatif d'un quelconque gene; procedes et produits connexes
WO1999063077A3 (fr) * 1998-06-04 2000-06-29 Tm Technologies Inc Compositions d'acide nucleique modifiant les caracteristiques de liaison d'un ligand; procedes et produits connexes

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5593834A (en) * 1993-06-17 1997-01-14 The Research Foundation Of State University Of New York Method of preparing DNA sequences with known ligand binding characteristics

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US5593834A (en) * 1993-06-17 1997-01-14 The Research Foundation Of State University Of New York Method of preparing DNA sequences with known ligand binding characteristics

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BLACKWELL T K ET AL: "DIFFERENCES AND SIMILARITIES IN DNA-BINDING PREFERENCES OF MYOD AND E2A PROTEIN COMPLEXES REVEALED BY BINDING SITE SELECTION", SCIENCE, vol. 250, no. 4984, 23 November 1990 (1990-11-23), pages 1104 - 1110, XP000371689 *
OLIPHANT A R ET AL: "DEFINING THE SEQUENCE SPECIFICITY OF DNA-BINDING PROTEINS BY SELECTING BINDING SITES FROM RANDOM-SEQUENCE OLIGONUCLEOTIDES: ANALYSIS OF YEAST GCN4 PROTEIN", MOLECULAR AND CELLULAR BIOLOGY, vol. 9, no. 7, July 1989 (1989-07-01), pages 2944 - 2949, XP000673592 *
PIERROU S ET AL: "SELECTION OF HIGH-AFFINITY BINDING SITES FOR SEQUENCE-SPECIFIC, DNABINDING PROTEINS FROM RANDOM SEQUENCE OLIGONUCLEOTIDES", ANALYTICAL BIOCHEMISTRY, vol. 229, no. 1, 20 July 1995 (1995-07-20), pages 99 - 105, XP000524716 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042621A3 (fr) * 1998-02-21 2000-03-16 Tm Technologies Inc Proprietes thermodynamiques d'acides nucleiques
WO1999063117A1 (fr) * 1998-06-04 1999-12-09 Tm Technologies, Inc. Procede de creation de sequences polynucleotidiques flanquantes qui vehiculent des affinites de liaison relatives a un site de liaison de ligand
WO1999063074A3 (fr) * 1998-06-04 2000-04-06 Tm Technologies Inc Methode permettant de modifier le niveau d'expression relatif d'un quelconque gene; procedes et produits connexes
WO1999063077A3 (fr) * 1998-06-04 2000-06-29 Tm Technologies Inc Compositions d'acide nucleique modifiant les caracteristiques de liaison d'un ligand; procedes et produits connexes

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EP1042510A1 (fr) 2000-10-11
JP2001526905A (ja) 2001-12-25

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