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WO1999063117A1 - Procede de creation de sequences polynucleotidiques flanquantes qui vehiculent des affinites de liaison relatives a un site de liaison de ligand - Google Patents

Procede de creation de sequences polynucleotidiques flanquantes qui vehiculent des affinites de liaison relatives a un site de liaison de ligand Download PDF

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Publication number
WO1999063117A1
WO1999063117A1 PCT/US1999/012558 US9912558W WO9963117A1 WO 1999063117 A1 WO1999063117 A1 WO 1999063117A1 US 9912558 W US9912558 W US 9912558W WO 9963117 A1 WO9963117 A1 WO 9963117A1
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WIPO (PCT)
Prior art keywords
molecules
ligand
duplex
binding site
nucleic acid
Prior art date
Application number
PCT/US1999/012558
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English (en)
Inventor
Michael J. Lane
Albert S. Benight
Brian D. Faldasz
Original Assignee
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.)
Filing date
Publication date
Application filed by Tm Technologies, Inc. filed Critical Tm Technologies, Inc.
Publication of WO1999063117A1 publication Critical patent/WO1999063117A1/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

  • 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. 0 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) form as opposed to the normal right-handed B form in the presence of a high salt environment (Pohl, FM and Jovin, TM (1972) J Mol. Biol, 67:375-396).
  • DNA sequence information has been and is being accumulated at an ever-increasing rate.
  • extant coding (and non-coding) DNA sequences it is still unclear how to translate DNA sequence information so as to reveal which segments of DNA in a given string will be more reactive to DNA binding agents in a DNA sequence of interest. o Although the function of genomic repetitive DNA sequences is not fully understood it is clear that these sequences are highly abundant and are quite capable of influencing the pattern of cellular gene expression.
  • the present invention relates to an efficient and economical method for creating and thereby systematically sampling different polynucleotide sequences of any given length sequence space for polynucleotide sequences given the nucleotides A, C, G, and T.
  • a plurality of different polynucleotide sequences of any specified length sequence space are created that represent polynucleotide sequences utilizing the four nucleotides A, C, G, and T.
  • the plurality of sequences may be randomly synthesized but can include any predetermined combination of the nucleotides A, G, C, or T.
  • a plurality of different polynucleotide sequences of any specified length sequence space are created that represent polynucleotide sequences utilizing the four nucleotides A, C, G, and T, and wherein the sequences are further modified chemically, for example by a methyl transferase.
  • the plurality of sequences may be randomly synthesized but can include any predetermined combination of the nucleotides A, G, C, or T.
  • the restriction endonuclease BamHl is added to a population of duplex polynucleotide molecules wherein the duplex polynucleotide molecules comprise a BamHl binding site (5'-GGATCC-3') flanked on either side or adjacent to a stretch of nucleotides under conditions which allow BamHl to contact its 5 binding site but not cleave it. Under these conditions only a fraction of the population of duplex polynucleotide molecules will bind to BamHl.
  • the bound subpopulation of duplex polynucleotide molecules can be separated from the unbound subpopulation of duplex polynucleotide molecules by methods well known to those skilled in the art.
  • the restriction endonucleases Mspl or o Hp ⁇ ll are added to a population of duplex polynucleotide molecules wherein the duplex polynucleotide molecules comprise a methylated (5'-C m CGG-3') or unmethylated (5'-CCGG-3') Mspl and Hpall binding site flanked on either side or adjacent to a stretch of polynucleotides under conditions which allow Mspl or Hpall to contact its binding site but not cleave it. Under these conditions only a fraction of the duplex s polynucleotide molecules will bind to Mspl or Hpall.
  • the bound duplex polynucleotide molecules can be separated from the unbound subpopulation of duplex polynucleotide molecules by methods well known to those skilled in the art.
  • the enzyme Hp ll-methylase is added to a population of polynucleotide molecules which are ligated into a synthetic DNA construct directly o flanking or adjacent to a polynucleotide duplex ligand binding site comprising of the sequence 5'-CCGG-3'.
  • the restriction endonucleases Mspl or ⁇ pall are subsequently added under conditions which allow Mspl or Hpall to contact its binding site but not cleave it. Under these conditions only a fraction of the population of duplex polynucleotide molecules will bind to Mspl or Hpall.
  • the bound subpopulation of 5 duplex polynucleotide molecules can be separated from the unbound subpopulation of duplex polynucleotide molecules by methods well known to those skilled in the art.
  • a subpopulation of polynucleotide sequences are ranked into a range of relative binding affinities from high binding affinity to low binding affinity, with respect to conferring a given relative binding affinity of any DNA ligand to o its binding site placed adjacent to a polynucleotide sequence based on its nucleotide composition.
  • FIG. 1 shows a schematic representation of the method of creating and grading into a range of relative binding affinities from high binding affinity to low binding affinity subsets of a random polynucleotide sequence flanking a BamHl binding site 5 based on the ability of the nucleotide composition of the subsets of flanking polynucleotide sequences to confer relative binding affinity for BamHl binding to its binding site.
  • polynucleotide includes 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 both polyribonucleotides and 5 polydeoxynbonucleotides. Polynucleotides can be obtained from existing nucleic acid sources (e.g., genomic DNA or cDNA), but can also be synthetic (e. g., produced by oli
  • a "ligand” includes any chemical moiety selected from the group consisting of: a compound which binds to a duplex polynucleotide in a sequence-specific way; a o compound which binds to a duplex polynucleotide sequence in a non-specific way; a protein; an enzyme; an enzyme which alters the structure of a duplex polynucleotide sequence to which it binds; an enzyme which alters the structure of a duplex polynucleotide sequence to which it binds by breaking or forming a covalent or non-covalent bond between an atom of the nucleic acid and another atom; an enzyme 5 which cleaves one or both strands of a duplex polynucleotide sequence to which it binds; a restriction enzyme; a restriction endonuclease; an enzyme which methylates a duplex polynucleotide sequence to which it binds; an enzyme which alkylates a duplex polynucle
  • a “ligand binding site” or “binding site” includes any domain or subdomain in a nucleic acid molecule which directly contacts a ligand by hydrogen bonding, van der Waals radius interactions, and/or electron cloud interaction with the bases of a nucleic acid molecule, or indirectly via a salt or water molecule.
  • a “flanking sequence” is a polynucleotide sequence located adjacent a ligand binding site of a nucleic acid molecule.
  • "relative binding affinities" of nucleotide flanking sequences are measured for different nucleic acid molecules in which different flanking sequences are located adjacent the same ligand binding side.
  • the first molecule is said to have a binding affinity that is "relatively higher” than the binding affinity of the second molecule.
  • An endonuclease is said to be "substantially free of cleavage activity" when 5 under the given conditions, there is substantially no observable cleavage.
  • a “pure repeat” is a repeating DNA sequence for which all base positions are defined. For example, if the nucleotides in a pure di-nucleotide repeat are A and G, then a pure dinucleotide repeat is (AG) n where n is the number of times (AG) is repeated. Similarly, if the nucleotides in a trinucleotide repeat are A, G, and C, then a pure o trinucleotide repeat is (AGC) n where n is the number of times (AGC) is repeated.
  • the definition is not intended to be limiting to dinucleotide or trinucleotide repeats and can be extended to tetranucleotide repeats, pentanucleotide repeats, and higher repeating units.
  • an "impure repeat” is a repeating DNA sequence for which one or more base positions allows for the insertion of a random nucleotide. For example, if one of the nucleotides in an impure dinucleotide repeat is A and the random nucleotide is X where
  • X is either A, C, G, or T
  • an impure di-nucleotide repeat is 5'-(AX) n -3' where n is the number of times (AX) is repeated.
  • a and G the third random nucleotide is X
  • X is either A, C, G, or T
  • an impure trinucleotide repeat is 5'-(AGX) n -3' where n is the number of times (AGX) is repeated.
  • This definition is not intended to be limiting to dinucleotide or trinucleotide repeats and can be extended tetranucleotide repeats, pentanucleotide repeats, and higher repeating units.
  • a "family" of DNA sequences is a group of DNA sequences that are related by virtue of their conforming to the rules defined herein for the sequence design and synthesis.
  • a "frame" of a DNA repeat refers to the minimum successively repeating DNA sequence or motif in a DNA sequence. For example, one frame in the sequence
  • 5'-GCGCGC-3' would be GC. In the sequence 5'-GCTGCTGCT-3', one frame would be
  • An "initial frame" of a DNA repeat refers to the minimum successively repeating
  • the initial frame in the sequence 5'-GCGCGC-3' would be GC.
  • 5'-GCTGCTGCT-3' would be GCT.
  • a "shifted frame” or “frame shifting” is any of the unique, non-initial frames in a repeating DNA sequence or motif.
  • a shifted frame is CG.
  • a shifted frame is CTG.
  • the present invention is illustrated by the following examples relating to the creation of duplex polynucleotide flanking sequences and measurements which affect relative binding affinities of a binding site flanked by these polynucleotide sequences.
  • Example 1 illustrates schematically the method of the present invention.
  • Example 2 describes the creation of families of pure repeat sequences of specified length.
  • Example 3 describes the creation of families of impure repeat sequences of specified length.
  • EXAMPLE 1 Schematic of the method of the present invention Shown in the box at the top of Figure 1 are three DNA constructs which will serve as examples. Each construct consists of (from left to right, 5' - 3') a unique PCR primer site followed by a repeat motif of length n; the nucleotide adenosine (A); a BamHl binding site; a second adenosine (A) residue followed by another repeat motif of length n; and finally a second unique PCR primer site. Each primer site includes a unique restriction endonuclease cleavage sequence.
  • Construct 1 designated (AX) n , contains n repeating dinucleotide motifs of the base A followed by a random base.
  • (GX) ⁇ contains n repeating motifs of the base G followed by a random base.
  • (XX) n contains n repeating motifs that are completely random.
  • the constructs are synthesized as single-stranded polynucleotide molecules by methods well known in the art. The single-stranded polynucleotide constructs are subsequently "filled-in” by using either the PCR, reverse transcriptase, or the Klenow fragment of DNA polymerase I, resulting in duplex 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 BamHl while some of the duplexes are not bound.
  • Those sequences which bind BamHl with a relatively high affinity bind the endonuclease in preference to those sequences which bind BamHl 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 BamHl concentrations. The conditions of the experiment are such that BamHl contacts the binding site but does not cleave it.
  • binding affinity for each subpopulation can then be determined by means known in the art, for example, by plotting the fraction of duplexes bound versus the BamHl concentration as shown in the figure at the bottom.
  • sequence motifs of the 5 desired repeat length i.e., the initial frame are created, for example, by execution of a computer program, by iterating the four possible base substitutions in each base position of the repeating sequence.
  • sequence motifs In the trivial case of a single-base repeat, with a repeat length of 1, there are four possible sequence motifs: (A) n , (C) n , (G) n , and (T) w .
  • the di-nucleotide repeat motifs also include the single-nucleotide repeats.
  • sequence motifs of the desired repeat length i.e., the initial frame
  • sequence motifs of the desired repeat length are created, for example, by execution of a computer program, by iterating the four possible base substitutions in each base position of the repeating sequence.
  • a dinucleotide repeat with a repeat length of 2
  • sequence motifs there are 16 possible sequence motifs: (AA) n , (AC) n , (AG) W , (AT) ⁇ , (GA) precede, (GC) thread, (GG) technically, (GT) thread, (CA) thread, (CG) wool, (CC) thread, (CT) thread, (TA) thread, (TC) combat, (TG) combat, (TT) ⁇ .
  • each of these sequence motifs the last base position of each frame is replaced with a nucleotide "X,” indicating that the base position may be any base permitted by the DNA synthesis process.
  • X nucleotide
  • the following impure repeats result from this step: (AX) n , (CX) n , (GX) ⁇ , (TX) «.
  • a tri-nucleotide repeat there are 64 possible sequence motifs:
  • the unique sequences are examined and grouped together into families according to equivalence by complementarity, for example by execution of a computer program. Since duplex DNA is assumed, the impure dinucleotide repeat motif (AX) n is equivalent to the motif (XT) n by complementarity.
  • the resulting impure sequence motifs are synthesized by means known in the art, with the base positions denoted "X” synthesized with the appropriate base nucleotides to generate populations of oligonucleotides representative of sequences matching the motif, e.g., by permitting all four bases into the synthesis reaction at the steps corresponding to the "X" base positions in the polynucleotide sequence.
  • dinucleotide repeats illustrate a method for creating six families of sequence motifs which represent an efficient and economical means of synthesizing and characterizing the relative reactivities of polynucleotide sequences from the sixteen individual motifs that would have to be synthesized and characterized if the method of this invention were not employed.
  • trinucleotide repeats sixty-four original sequence motifs can be reduced to just eight families of sequence motifs.
  • the utility of the above invention is not limited to repeat sequences illustrated above but can be extended to tetranucleotide repeats, pentanucleotide repeats, and higher repeating units.

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Abstract

Cette invention concerne un procédé qui permet de créer et par conséquent d'échantillonner de manière systématique des séquences polynucléotidiques différentes d'un espace de séquence de n'importe quelle longueur donnée pour des séquences polynucléotidiques contenant les nucléotides A, C, G et T. Les séquences peuvent ensuite être rangées sous forme d'un classement d'affinités de liaison relatives entre une forte affinité de liaison et une faible affinité de liaison, en rapport avec l'attribution d'une affinité de liaison relative donnée d'un ligand d'ADN quelconque à son site de liaison placé juste à côté d'une séquence polynucléotidique sur la base de sa composition nucléotidique.
PCT/US1999/012558 1998-06-04 1999-06-04 Procede de creation de sequences polynucleotidiques flanquantes qui vehiculent des affinites de liaison relatives a un site de liaison de ligand WO1999063117A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US8793598P 1998-06-04 1998-06-04
US60/087,935 1998-06-04
US9036098P 1998-06-23 1998-06-23
US60/090,360 1998-06-23
US32561499A 1999-06-03 1999-06-03
US09/325,614 1999-06-03

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063077A2 (fr) * 1998-06-04 1999-12-09 Tm Technologies, Inc. Compositions d'acide nucleique modifiant les caracteristiques de liaison d'un ligand; procedes et produits connexes
WO2001051669A2 (fr) * 2000-01-15 2001-07-19 Genelabs Technologies, Inc. Methode de dosage et de selection pour sites de liaison aux acides nucleiques
US6420109B1 (en) * 1998-09-11 2002-07-16 Genelabs Technologies, Inc. Nucleic acid ligand interaction assays
WO2002083894A1 (fr) * 2001-04-10 2002-10-24 Panbio Limited Sequence d'acide nucleique lineaire comprenant des repetitions et denuee de structure secondaire

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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
WO1999032664A1 (fr) * 1997-12-23 1999-07-01 Tm Technologies, Inc. Procede de selection de sequences adjacentes porteuses d'affinites de liaison relatives a un site de liaison aux ligands

Patent Citations (2)

* Cited by examiner, † Cited by third party
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
WO1999032664A1 (fr) * 1997-12-23 1999-07-01 Tm Technologies, Inc. Procede de selection de sequences adjacentes porteuses d'affinites de liaison relatives a un site de liaison aux ligands

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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, 1 July 1989 (1989-07-01), pages 2944 - 2949, XP000673592, ISSN: 0270-7306 *
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999063077A2 (fr) * 1998-06-04 1999-12-09 Tm Technologies, Inc. Compositions d'acide nucleique modifiant les caracteristiques de liaison d'un ligand; 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
US6420109B1 (en) * 1998-09-11 2002-07-16 Genelabs Technologies, Inc. Nucleic acid ligand interaction assays
WO2001051669A2 (fr) * 2000-01-15 2001-07-19 Genelabs Technologies, Inc. Methode de dosage et de selection pour sites de liaison aux acides nucleiques
WO2001051669A3 (fr) * 2000-01-15 2002-06-20 Genelabs Tech Inc Methode de dosage et de selection pour sites de liaison aux acides nucleiques
US6613517B2 (en) 2000-01-15 2003-09-02 Genelabs Technologies, Inc. Nucleic acid binding assay and selection method
WO2002083894A1 (fr) * 2001-04-10 2002-10-24 Panbio Limited Sequence d'acide nucleique lineaire comprenant des repetitions et denuee de structure secondaire

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