EP0549692A4 - Protein sequence-specific oligonucleotide sequences - Google Patents

Protein sequence-specific oligonucleotide sequences

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Publication number
EP0549692A4
EP0549692A4 EP19910917153 EP91917153A EP0549692A4 EP 0549692 A4 EP0549692 A4 EP 0549692A4 EP 19910917153 EP19910917153 EP 19910917153 EP 91917153 A EP91917153 A EP 91917153A EP 0549692 A4 EP0549692 A4 EP 0549692A4
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European Patent Office
Prior art keywords
oligonucleotide
sequence
mixture
sequences
complex
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EP19910917153
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EP0549692A1 (fr
Inventor
Harold Martin Weintraub
Thomas Keith Blackwell
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Fred Hutchinson Cancer Center
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Fred Hutchinson Cancer Research Center
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Publication of EP0549692A1 publication Critical patent/EP0549692A1/fr
Publication of EP0549692A4 publication Critical patent/EP0549692A4/en
Withdrawn legal-status Critical Current

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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

  • PROTEIN SEQUENCE-SPECIFIC OLIGONUCLEOTIDE SEQUENCES
  • the invention is directed to a method to identify oligonucleotide sequences which specifically bind target proteins. More specifically, it concerns a method to identify the appropriate oligonucleotide sequence for such binding, and several oligonucleotide sequences which correspond to proteins known to be instrumental in differentiation.
  • RNA oligonucleotides to their complements to inactivate such specific DNA or RNA oligonucleotides which mediate diseases or other undesirable conditions in humans, animals, and even plants.
  • the origin of the term "antisense” is thus clear: the therapeutic or diagnostic oligonucleotide would be the antisense counterpart of the targeted RNA or DNA.
  • the "antisense” oligonucleotides can be supplied directly or generated in situ and may either be conventional oligomers, or are, more commonly, oligomers having properties which make them, for example, resistant to nucleases, more capable of transfer across membranes, or more capable of specific binding to the desired target.
  • the oligonucleo- tides used in this approach may recognize double- stranded DNA by binding to the major or minor grooves present in the double-helix.
  • oligonucleotide-based therapy to include binding to duplexed DNA was made possible by elucidation of the rules governing sequence-specific binding in this context. While not so precisely under- stood as the requirements for base-pair complementation, these principles have been sufficiently described to make de novo design of oligomers which will bind to known target duplexes possible. Such de novo design of specifically binding oligonucleotides is not, however, possible with respect to non-oligonucleotide targets. Formulation of an approach that would permit construc ⁇ tion of oligonucleotides capable of specific binding to any desired target substance would clearly be desirable.
  • oligonucleotides By use of such oligonucleotides, the modulation of the metabolic events associated with any condition, disease, or developmental process for which any critical sub ⁇ stance is known could be effected. Furthermore, the specifically binding oligonucleotides are useful in diagnostic and assay methods and in regulation of cell cultures jln vitro. The method of the invention permits just such design of oligonucleotides comprising seguences specific for any target substance of sufficient size to show complexation with DNA or RNA sequences.
  • the invention method utilizes the polymerase chain reaction (PCR) technique, as described by Saiki, R.K. , et al., Science (1988) 239:487-491.
  • PCR polymerase chain reaction
  • Joyce, G.F. Gene (1989) 82.:83-87 applied the PCR reaction to plus strand RNA/minus strand DNA complexes to study the evolution of RNAs with catalytic activity.
  • Various strategies for producing mutations in RNA to provide the catalytic activity are discussed. Robertson, D.L. , and Joyce, G.F. , in a letter to Nature (1990) 344:467-468. describe the results of application of this technique to obtain a catalytic RNA which cleaves DNA more efficiently than the wild-type enzyme.
  • Randomly synthesized DNA yielding approximately 10 15 i.ndividual sequences was amplified by PCR and transcribed into RNA. It was thought that the complexity of the pool was reduced in the amplification/transcription steps to approximately 10 13 different sequences.
  • the pool was then applied to an affinity column containing the dye and the bound sequences subsequently eluted, treated with reverse transcriptase and amplified by PCR. The results showed that about one in 10 random sequence RNA molecules folds in such a way as to bind specifically to the ligand.
  • Tuerk, C. and Gold, L. in Science (1990) 249:505-510 used what they referred to as the procedure of "systematic evolution of ligands by exponential enrichment" (Selex) which is described as follows: a pool of RNAs that are completely randomized at specific positions is subjected to selection for binding to a desired protein which has been displayed on a nitrocellulose filter. The selected RNAs are then amplified as double-stranded DNA that is competent for subsequent in vitro transcription. The newly transcribed RNA is then enriched for better binding sequences and recycled through this procedure. The amplified selected sequences are subjected to sequence determination using dideoxy sequencing. Tuerk and Gold applied this procedure to determination of RNA ligands which bind to T4 DNA polymerase.
  • Thiesen, H.-J. and Bach, C. Nucleic Acids Res (1990) 18_:3203-3208 described what they call a target detection assay (TDA) to determine DNA binding sites for putative DNA binding proteins.
  • TDA target detection assay
  • a purified functionally active DNA binding protein and a pool of random double-stranded oligonucleotides which contain PCR primer sites at each end were incubated with the protein.
  • the resulting DNA complexes with the protein in their case, the SP1 regulatory protein
  • the invention herein utilizes a binding site selection technique which also depends on the avail ⁇ ability of PCR.
  • selected and ampli ⁇ fied binding sites provide a characteristic imprint of protein binding.
  • this process is aided by consensus sequences.
  • the invention is directed to a method to determine oligonucleotide sequences that specifically bind proteins or other targets.
  • the method is especially applicable to DNAs wherein a consensus sequence site is known. In this case, knowledge of the nature of the protein or other target which is bound is not necessarily a requisite.
  • This technique has been applied to describe the nucleotide sequences responsible for binding certain basic helix-loop-helix (bHLH) proteins which are important in differentiation, specifically MyoD, cMYC and a previously undescribed protein from reticulocytes.
  • bHLH basic helix-loop-helix
  • the invention is directed to a method to determine an oligonucleotide sequence which binds specifically to a target ligand, which method comprises providing a mixture containing oligomers having portions which form a random set of sequences and portions which permit amplification of the oligomers, treating the oligomer mixture with the target substance to form complexes between the target and the oligonucleotides bound specifically thereto, separating the complexes from the unbound members of the oligo- nucleotide mixture, recovering the complexed oligo ⁇ nucleotide(s) and amplifying these. This process will generally be repeated over several rounds of complexation, separation and amplification.
  • the mixture of oligo ⁇ nucleotides having random sequences also contains a consensus sequence known to bind the target.
  • the invention is directed to oligonucleotides identified by the above method, and to oligonucleotide sequences which bind specifically to MyoD, cMYC, and a bHLH protein from reticulocytes.
  • the invention is directed to complexes comprising target substance and specifically bound oligomer in a cell-free environment.
  • the invention is directed to oligomers which contain sequences that bind specifically to target substances, and to the use of these oligomers in therapy, diagnostics, and purification procedures.
  • Figure 1 shows a diagrammatic representation of the method of the invention.
  • Figure 2 shows the DNA sequences of four oligomers used to illustrate the method of the invention.
  • Figure 3 shows typical separation results on an electrophoretic mobility shift assay (EMSA) of free oligonucleotides and bound oligonucleotides to a MyoD- containing fusion protein.
  • ESA electrophoretic mobility shift assay
  • Figure 4 shows typical sequencing results obtained from a control and complexed oligonucleotide recovered from the gel of Figure 3.
  • Figure 5 shows the electrophoretic mobility separation (EMSA) of complexes formed by proteins obtained by in vitro transcription/translation with random and nonrandom oligonucleotide probes.
  • Figure 6 is a higher exposure of the EMSA results of Figure 5, along with a comparable exposure of an EMSA obtained from an additional complexation reaction.
  • ESA electrophoretic mobility separation
  • Figure 7 shows the results of EMSA separations of oligomers retrieved by the process of the invention after additional rounds of complexation, separation, amplification and recovery.
  • Figure 8 shows sequencing results of the control oligonucleotide mixture and various selected oligomers from the mixture obtained from the complexes shown in Figure 7.
  • Figure 9 is a summary of the sequences of oligonucleotides obtained by the selection process of the invention.
  • Figure 10 shows an EMSA separation of proteins from myoblast and MEL cell extracts complexed to oligomer selected using binding to a crude reticulocyte lysate.
  • Figure 11 shows EMSA results of recoveries after selection by method of the invention from randomized oligomers using cMYC fusion proteins.
  • Figure 12 shows the results of sequencing the recovered oligomers of Figure 11 after three rounds of selection.
  • the invention is directed to a method which permits the recovery and deduction of oligomeric sequences that bind specifically to desired targets, including proteins. Therefore, as a result of appli ⁇ cation of this method, oligonucleotides which contain the specifically binding sequences can be prepared and used in oligonucleotide-based therapy and in other applications. For example, these oligonucleotides can be used as a separation tool for retrieving the substances to which they specifically bind. By coupling the oligo ⁇ nucleotides containing the specifically binding sequences to a solid support, for example, proteins or other cellular components to which they bind can be recovered in useful quantities.
  • these oligonucleo ⁇ tides can be used in diagnosis by employing them in specific binding assays for the target substances.
  • the specifically binding oligonucleotides can also be used for in vivo imaging or histological analysis.
  • Oligonucleotides includes RNA or DNA sequences of more than one nucleotide in either single chain or duplex form and specifically includes short sequences such as dimers and trimers, in either single chain or duplex form, which may be intermediates in the production of the specifically binding oligonucleotides.
  • specifically binding oligonucleotides refers to oligonucleotides which are capable of forming complexes with an intended target substance in an environment wherein other substances in the same environment are not complexed to the oligonucleotide. In general, a minimum of approximately 10 nucleotides, preferably 15 nucleotides, are necessary to effect specific binding.
  • the specifically binding oligonucleotides need to contain the sequence- conferring specificity, but may be extended with flanking regions and otherwise derivatized.
  • the specifically binding oligonucleotides may be sequenced, and then resynthesized in any convenient form for the intended use.
  • the oligonucleotides identified by the method of the invention in effect can include modifications both to the backbone structure and to the bases substituted thereon that may confer desirable properties, such as enhanced permeation or increased stability with respect to nucleases.
  • the information obtained by analysis of the oligonucleotide pool obtained as a result of the invention process is thus used in synthesis of oligonucleotides with any desired modification.
  • the oligonucleotides that comprise the sequences specifically binding to target substance may be conventional DNA or RNA moieties, or may be "modified" oligomers which are those conventionally recognized in the art.
  • the oligomers of the invention are defined also to include intermediates in their synthesis, any of the hydroxyl groups ordinarily present may be protected by a standard protecting group, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5' or 3' terminal OH is conventionally activated; the alternate terminal 3' or 5 1 OH may be protected.
  • one or more phosphodiester linkages may be replaced by alternative linking groups.
  • alternative linking groups include, but are not limited to, embodiments wherein P(0)0 of the conventional phosphodiester is replaced by P(0)S, P(0)NR_, P(0)R, P(S)S, P(0)OR ! , CO, or CNR 2 , wherein R is H or alkyl (1-6C) and R' is alkyl (1-6C) ; in addition, this group may be attached to adjacent nucleotide through O or S. Not all linkages in the same oligomer need to be identical.
  • oligonu ⁇ cleotides that which incorporate analogous forms of purines and pyrimidines.
  • "Analogous" forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents.
  • An exemplary but not exhaustive list includes 4-acetylcytosine, 5-(carboxy- hydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, dihydrouracil, inosine, N6-iso- pentenyladenine, 1-methyladenine, l-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil, beta-D-mannosylqueo
  • the oligonucleotides containing the specific binding sequences discerned through the method of the invention can also be derivatized in various ways. For example, if the oligonucleotide containing the specifically binding sequence is to be used for separ ⁇ ation of the target substance, conventionally the oligo- nucleotide will be derivatized to a solid support to permit chromatographic separation. If the oligonucleo ⁇ tide is to be used to label cellular components or other ⁇ wise for attaching a detectable moiety to target, the oligonucleotide will be derivatized to include a radio- nuclide, a fluorescent molecule, a chromophore or the like.
  • the oligonucleotide is to be used in specific binding assays, coupling to solid support or detectable label, and the like are also desirable. If to be used in therapy, the oligonucleotide may be derivatized to include ligands which permit easier transit of cellular barriers, toxic moieties which aid in the therapeutic effect, or enzymatic activities which perform desired functions at the targeted site. The oligonucleotide may also be included in a suitable expression system to provide for in situ generation of the desired sequence. In general, the oligonucleotides identified according to the method of the invention, and, if desired, synthesized de novo either in native or modified form are useful in a manner analogous to antibodies or specifically immunoreactive fragments thereof.
  • oligonucleotides are characterized by their ability specifically to bind the intended target molecule in both simple and complex environments.
  • the formation of an oligonucleotide-target complex may be formatted in procedures analogous to those employed in immunoassay procedures.
  • a wide range of such protocols is known in the art, and includes both direct and competitive formats, and involves employment of a wide range of detection techniques.
  • antibodies may be used in diagnostic and therapeutic applications, as well as in the control of cell growth and differentiation, so too may the oligonucleotides of the invention.
  • the oligonucleotides used as starting materials in the process of the invention to determine specific binding sequences may be single-stranded or double- stranded DNA or may be RNA. Double-stranded DNA is preferred.
  • the starting material oligo ⁇ nucleotide will contain a randomized sequence portion flanked by primer sequences which permit the application of the polymerase chain reaction to the recovered oligo ⁇ nucleotide from the complex. These flanking sequences may also contain other convenient features such as -13-
  • the randomized portion may be constructed usi g conventional solid phase techniques using mixtures of nucleotides at the positions where randomization is desired.
  • any degree of randomization may ise employed; some positions may be randomized by mixtures o.f only two or three bases rather than the conventional four; randomized positions may alternate with those wfeicfe have been specified. Indeed, it is helpful if some portions of the candidate randomized sequence are in frac known.
  • the target substances are proteins for which consensus sequences are known. While the method of the invention is illustrated using proteins as target substances, any ligand which is of sufficient size to be specifically recognized by an oligonucleotide sequence can be used, as the targe .
  • glycoproteins r proteins carbo- hydrates, membrane structures, receptors, lipids, organelles, and the like can be used as the complexafclr ⁇ i targets.
  • the process is greatly aided if a consensus sequence for the target is known.
  • a particular illustration of this application i, ⁇ set forth in the examples below with respect to the basic-HLH domains which characterize a number of pr ⁇ iteinis involved in development and differentiation of tissu&s.
  • These proteins include a region of basic amino acids which are followed in sequence (N ⁇ C) by a helix-loop— helix region which is thought to mediate multimerizafci ⁇ c of the proteins. The multimerization results in positioning the basic regions so as to make specific contacts with the DNA.
  • DNA sequences wkic ' fc bind proteins containing bHLH regions contain a palindromic consensus region CANNTG. Proteins containing the bHLH region are produced by the gene E2A, MyoD (which is associated with myogenesis and expression of muscle- specific genes) , cMYC (an oncogene) , and other genes involved in development described below.
  • E2A the gene that is associated with myogenesis and expression of muscle- specific genes
  • cMYC an oncogene
  • FIG. 1 An outline of the procedure of the invention is shown in Figure 1. The steps of this process result in "Selected and Amplified Binding-Sites" (SaABs) .
  • SaABs Select and Amplified Binding-Sites
  • a mixture of oligonucleotides is synthesized with random sequences in the intended binding site that are flanked by suitable regions for hybridization to primers for use in PCR.
  • item 1 a single strand DNA is prepared with random nucleotide sequence NNN where the region for primer hybridization,
  • A is shown at the 3' end.
  • the oligomer is formed into a duplex by synthesizing the opposite strand, which now has primer hybridization regions A and B. This is incubated with the target, in this case a protein, and the complexes shown as item 3 are separated from the uncomplexed duplexes using the mobility shift in electro- phoresis (EMSA) .
  • the bound templates are rescued by PCR and amplified for sequencing.
  • the original double- stranded oligonucleotide in item 2 is also amplified as a control.
  • the resulting amplified sequences are applied to sequencing gels to determine the nature of the "ABC" counterparts of the random nucleotides selected.
  • the entire process is repeated using the recovered and amplified duplex until sufficient resolution is obtained.
  • the procedure shown in Figure 1 is merely illustrative.
  • the mixture of oligonucleotides may be comprised of single-stranded DNA or RNA as well as the double-stranded DNA shown.
  • the primer sequences flank the randomized portion on a single oligonucleotide chain.
  • the separation of the portion of the mixture which binds to target substance may be conducted in any convenient manner. For example, rather than relying on a difference in electrophoretic mobility of the complex as compared to the unbound oligonucleotide, the target substance may be coupled with a solid support and the oligonucleotide mixture applied to the support.
  • the procedure simply involves complexation of the mixture with the target, separation of the complexed oligonucleotides from those failing to participate in the complex and rescue of the complexed oligonucleotides by amplification.
  • the amplification of the oligonucleotides which bind to the target may be conducted either while the complex is still intact or after prior separation of the complexed oligonucleotides from the target. In general, more than one "round" of binding, separation of the complex, and amplification will be required in order to achieve a set of appropriately binding oligonucleotides.
  • the process is simply repeated using the recovered subset of binding nucleotides as starting material in subsequent rounds until a mixture containing sufficient binding affinity is obtained.
  • this specifically binding subset it will be desirable to sequence this specifically binding subset to determine consensus sequences in the specifically binding oligomers.
  • the members of this subset may then be synthesized de novo, thus permitting the preparation of oligonucleotides that contain either base modifications, backbone modifications, or both.
  • the oligomers of the invention which contain specifically binding nucleotide seguences are useful in therapeutic, diagnostic and research contexts.
  • the oligomers are utilized in a manner appropriate for oligonucleotide therapy in general—as described above, oligonucleotide therapy as used herein includes any use of oligonu ⁇ cleotides as medicaments, whether this involves targeting a specific DNA or RNA or targeting any other substance through complementarity or through any other specific binding means, for example, sequence-specific orientation in the major groove of the DNA double-helix, or any other specific binding mode.
  • the oligomers of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration.
  • the oligomers of the invention are formulated in liquid solu ⁇ tions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addi ⁇ tion, the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays, for example, or using suppositories.
  • the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the oligonucleotides may also be- employed in expression systems, which are administered according to techniques applicable, for instance, in applying gene therapy.
  • the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target substances to which they specifically bind. Such diagnostic tests are conducted by contacting a sample with the specifically binding oligonucleotide to obtain a complex which is then detected by conventional means.
  • the oligomers may be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support to which the target substance has been bound through a specific or nonspecific binding means detected.
  • the specifically binding oligomers may be used to effect initial complexation to the support. Means for conducting assays using such oligomers as specific binding partners are generally known to track those for standard specific binding partner based assays.
  • oligomers of the invention are characterized by their ability to target specific substances regardless of the mechanisms of targeting or the mechanism of the effect thereof.
  • the specifically binding oligonucleotides of the invention are especially helpful in effecting the isolation and purification of substances to which they bind.
  • the oligonucleotide containing the specific binding sequences is conjugated to a solid support and used as an affinity ligand in chromatographic separation of the target substance.
  • the affinity ligand can also be used to recover previously unknown substances from sources which do not contain the target substance by virtue of binding similarity between the intended target and the unknown proteins.
  • insight may be gained as to the mechanisms for control of gene expression.
  • oligonucleotide sequences in the randomized mixtures were synthesized using standard solid-phase synthesis techniques and are shown in Figure 2.
  • the MCK muscle creatine kinase enhancer
  • Oligomers DI, D2 and D3 have various locations of randomization of sequence, and further contain regions for coupling to PCR primers shown as B and A" at the 5' and 3' ends, respectively.
  • Primer A is 5 • -TCCGAATTCCTACAG-3 * and primer B is
  • the double-stranded D1-D3 templates were generated by annealing the oligonucleotide to. a 10-fold molar excess of primer A, synthesizing the complementary strand using Klenow fragment of E. coli DNA polymerase and purifying the template on a 12% polyacrylamide gel.
  • the templates were end-labeled using the kinase reaction of Davis, R.L., et al., Cell (1990) 60:733.
  • the MCK double- stranded template was obtained from a kinased oligo ⁇ nucleotide annealed to its complement.
  • the randomization obliterates a portion of the consensus sequence in each case.
  • randomization is limited to two nucleotides upstream, two nucleotides downstream, and the two nucleotides between the members of the consensus motif.
  • a slice approximately 0.3 cm wide was excised from the dried- down gel including the 3 MM (Whatman) paper backing.
  • the gel slices were incubated at 37 ⁇ C overnight in 0.5 ml of 0.5 M ammonium acetate, 10 mM MgCl, 1 mM EDTA, and 0.1% SDS. Approximately 50% of the radioactivity was recovered.
  • the eluate was extracted twice each with phenol and with chloroform:isoamyl alcohol, 24:1, and precipitated with ethanol. The precipitates were brought to 0.3 M sodium acetate and reprecipitated with ethanol.
  • the recovered and amplified complexed oligomers were then sequenced using labeled primer A or B and the termination step of the Sequenase procedure marketed by United States Biochemical Co. as follows.
  • the primers were labeled using a kinase reaction to 1-2x10 cpm/ng and unincorporated label was removed using a Sephadex G50 spin column. 10 ng labeled primer were mixed with about 5 ng purified oligomer to be sequenced in a 12 ⁇ l reaction that contained 1 ⁇ l Sequenase Mn +2 buffer and 2 ⁇ l 5 x Sequenase buffer. The reaction was incubated at 95°C for 5 min and then quick spun at room temperature for 1 min.
  • the reaction was placed on ice and to it were added 1 ⁇ l 0.1 M dithiothreitol and 2 ⁇ l of diluted Sequenase 2.0 enzyme (1:8 in ice-cold TE, pH 7.4). 3.5 ⁇ l of this mixture were added to 2.5 ⁇ l of each of the Sequenase dGTP termination mixes and incubated at 45°C for 4 min. The reactions were terminated by adding 4 ⁇ l
  • Example 2 DNA Sequences Targeting Various Proteins
  • the D3 oligomer which contains this sequence was used in subsequent studies. While D3 contains the consensus sequence, it is randomized in the immediate proximity.
  • Various proteins which are associated with differentiation including MyoD, E2A, E12, and E47, were synthesized by in vitro translation from DNAs, some of which are reported by Murre, C, et al., Cell (1989) jjj5:777. The transcribed sequences were prepared from a mouse MyoD cDNA, a human E12 cDNA (E12R) and a human E47 cDNA as described by Benezra, R.
  • the final binding reaction to test randomized oligomers contained 20 mm Hepes, pH 7.6, 50 mM KC1, 1 mM dithiothreitol, 1 mM EDTA, 8% glycerol, 0.1 ⁇ g polydl/dC and 2 ⁇ g of a 50 bp single-stranded oligonucleotide, both added as nonspecific competitors.
  • Each binding reaction contained the in vitro synthesized protein species at about 6.9 x 10 M and either 0.15 ng of MCK or 0.30 ng of D2 or D3 labeled templates providing a protein:DNA molar ratio of about 0.18. Binding reactions were performed at room temperature for 20 min and immediately subjected to EMSA. The results of application of these incubation mixtures to EMSA are shown in Figure 5. These results indicate that MCK binds strongly .to E12/MyoD, E47 and E47/MyoD; D3 binds only to E47.
  • Figure 8 shows the results of sequencing performed as described above with some of the complexes shown in Figure 7 which were excised from the gel.
  • Figure 8A gives the results for the D3 control mixture showing complete heterogeneity at the six positions which were randomized.
  • DNA sequences which had been selected by the invention process ( Figures 8B-8F) showed positional preferences.
  • the D3 oligomer selected by complexation to MyoD ( Figure 8B) showed a clear preference for T in positions 5 and 4, retained some heterogeneity in positions 1 and -1, and showed some preference for A in positions -4 and -5.
  • the reticulocyte lysate shown in Figure 8F apparently recognizes the sequence (G/A)CCAGTTG(N)A.
  • Figure 9 A summary of the results of the binding and sequencing experiments illustrated in Figure 8 is shown in Figure 9.
  • the preset position choices are shown on shaded backing, the assignment preferences that are absolute or nearly so are indicated with capital letters, and incomplete preferences are printed in lower case. However, a bar over the letter indicates exactly the opposite—the base is never found at the indicated position (capitals) or only weakly represented (lower case) .
  • MCK and the lysate-derived D3 template were used in complexation reactions, conducted as described above under the following conditions: P2 myoblast binding reactions contain 20 mM Hepes, pH 7.6, 1.5 mM MgCl 2 , 50 mM NaCl, 5% glycerol and 500 ng poly(dI/dC) .
  • MEL cell binding reactions were conducted as described in Example 1, except that each reaction contained 2 ⁇ g of polydl/dC. Both binding reactions were incubated at room temperature for 20 minutes and then immediately subjected to EMSA on 5% polyacrylamide gels at 200 V at 4°C. The results are shown in Figure 10.
  • the lysate- selected D3 binds to factors in the MEL extract; and to different factors from that bound by MCK in the myoblast cell extract. These previously unidentified target proteins are therefore recoverable by virtue of their ability to bind lysate-selected D3.
  • Amplified D3 was used as a control. As indicated in the figure, the two bases internal to the consensus sequence have been identified, but heterogeneity in the flanking sequences persists.

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Abstract

Méthode servant à identifier des séquences d'oligomères qui se lient spécifiquement à des protéines cibles ou à d'autres fractions cibles. La technique comprend la complexation de la substance cible avec un mélange d'oligonucléotides contenant des séquences aléatoires et des séquences qui servent d'amorces pour des réactions en chaîne de polymérases dans des conditions dans lesquelles un complexe se forme avec certains éléments, mais pas tous, du mélange d'oligonucléotides. Le mélange est ensuite séparé des oligonucléotides non complexés et les éléments complexés du mélange d'oligonucléotides sont récupérés du complexe séparé et amplifiés en utilisant la réaction en chaîne de polymérase. On peut procéder à plusieurs tours de sélection en utilisant la complexation, la séparation, l'amplification et la récupération. Le mélange ou les oligonucléotides récupérés peuvent être mis en séquences et cette information peut être utilisée pour la synthèse de novo du(des) oligonucléotide(s) identifié(s). Ces oligonucléotides synthétisés peuvent contenir une ou plusieurs bases ou liaisons de squelette modifiées.
EP19910917153 1990-09-21 1991-09-18 Protein sequence-specific oligonucleotide sequences Withdrawn EP0549692A4 (en)

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