JPH06503715A - Protein sequence specific oligonucleotide sequences - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Nbaku 1 Oligonucleotide 1 technique 1L Kuno The present invention identifies oligonucleotide sequences that specifically bind to target proteins. Regarding how to More specifically, oligonucleotides suitable for such conjugation sequences and several ori- nes corresponding to proteins known to aid in differentiation. The present invention relates to a method for identifying oligonucleotide sequences.

"and ' A range of what are originally called "antisense" treatments and diagnostics has expanded over the past few years. It has been greatly expanded. The original concept was that the disease was found in humans, animals and even plants. specific RNAs or RNAs that mediate disease or other undesirable symptoms. DNA and RNA oligonucleotides to inactivate the A oligonucleotides. Let's take advantage of the specific hybridization genes for their complements It was intended to be.

The origin of the term "antisense" is clear as follows. i.e. treatment or diagnosis Antisense oligonucleotides can be used to target RNA or DNA antisense. Being a scar partner. “Antisense” oligonucleotides , which can be directly supplied or produced in 5 situ, using conventional sesame, or more generally, resistant to nucleases, e.g. properties such as the ability to cross membranes or bind more specifically to the desired target. It can be an oligonucleotide with However, the oligonucleotides used in this attempt In addition to specific binding influenced by conventional base pairing, cleotides are Can recognize double-stranded DNA by binding to the existing major groove or minor groove .

Such studies have investigated, for example, It has been suggested that it binds to promoter sequences and prevents transcription. Therefore, This concept goes beyond simple "antisense" research and involves the administration of oligonucleotides, or in 5 situ generation of oligonucleotides. It was expanded to include Construction of various oligomers useful for “antisense” therapy A general study on chnlues (1988) i:95g-976 and 5Lein, C,A , et al., Cancer Res (19811) 48:2659-2668. It has been praised. These are incorporated herein by reference.

Expansion of oligonucleotide-based therapies to include binding to double-stranded DNA , by elucidating the laws governing sequence-specific binding in this situation. It's now possible. These principles are clearly necessary for base pair complementarity. Although poorly understood, there are well-described and well-known molecules that bind to This enables novel designs of ligomers.

However, novel designs of such specifically binding oligonucleotides This is not possible for targets that are not oligonucleotides. Specific to any desired target substance The systematization of research that allows the construction of oligonucleotides that can bind differently is clearly It is desired by crabs. The use of such oligonucleotides allows the release of critical substances. Regulation of metabolic events associated with any known condition, disease, or developmental process It could have been done. Furthermore, specifically binding oligonucleotides are useful for diagnostic and It is useful in the cell culture method as well as in the regulation of cell culture in vitro. Inventor The method is just a DNA or RNA sequence that is large enough to be complexed with the DNA or RNA sequence. It is possible to design oligonucleotides with specific sequences for any target substance. make it possible.

The method of the present invention is described in 5aiki, R., et al., 5science (1988). 239:487-491 using the replication chain reaction (PCR) method. .

There are many related publications describing the use of this method in similar situations. For example, J. oyee, G.F., Gene (1989) 82:83-87 is a catalyst Positive-strand PCR reactions to study the evolution of active R)JA It is applied to RNA/minus-strand DNA complexes. Provides catalytic activity Various strategies have been considered for mutating RNA to serve the needs of humans. R obertson, D.L., and Joyce, G.F., Nature. (1990) 344:467-468. The results of applying this method to obtain catalytic RNA that efficiently cleaves DNA are as follows. Are listed.

Kinzler, N., W. et al., Nucleic Ac1ds Res (198 9) 17:3645-3653 binds to proteins that regulate gene expression. This method has been applied to identify DNA sequences that The report includes whole genome D NA is first converted into a form suitable for amplification by PCH, and the desired DNA sequence is selected by binding to the target regulatory protein. Then the recovered join Sequences are amplified by PCH. Selection and amplification steps are repeated as necessary. be done. As described, this step involves the transcription factor 3A was applied to the identification of DNA sequences that bind to The most recent work by the same authors (Kinzler et al.) Recent papers! 1101 Celt Biol (1990) 10:634-6 In 42, this same method was applied to GL produced as a recombinant fusion protein. It was applied to identify parts of the human genome that bind to I gene products. This GL The I gene is amplified in a subset of human tumors.

Ellington, A. D. et al., Nature (1990) 346:81 8-822 produces large numbers of randomly sequenced RNA molecules and small ligands. , in this paper, specific dyes such as C1bacron blue, describes the identification of these molecules that bind to Approximately 1015 individual arrangements Randomly synthesized DNA giving a sequence is amplified by PCH and transcribed into RNA. It was done. The degree of complexation of this pool is approximately 1013 in the amplification/transcription process. Columns were reduced. This pool is then applied to an affinity column containing the dye. the bound sequences are sequentially eluted, treated with reverse transcriptase, and subjected to PCR. It was more amplified. 1 out of 1010 randomly sequenced RNA molecules It was shown to fold in such a way that it binds specifically.

Tuerk, C., and Gold, L. 5science (1990) 2 49+505-510, where they ``exponentially increase the number of ligands. The method of systematic evolution J (5elex) was used. This is as below Are listed. A pool of RNA completely randomized at a specific position is It was subjected to selection for binding to the desired protein developed on cellulose. next , amplify the selected RNA as double-stranded DNA suitable for subsequent in vitro transcription. Ru.

The newly transcribed RNA is then multiplied for better binding sequences, and this process be used repeatedly in The amplified selected sequences are analyzed by dideoxy sequencing. sequence. Tuerk and Gold applied this method to T4 DNA polypeptides. It was applied to the measurement of RNA ligands that bind to merase.

Th1esen, H,-J, and Bach, C, Nucleic Ac1 ds Res (1990') 18:3203-3208 is the estimated DN They used target detection assays ( described an assay called TDA). In their research, purified functionally active a random NA-binding protein and a PCR primer site at each end. A pool of double-stranded oligonucleotides was incubated with the protein. profit DNA complex with this protein (in their case, 5PIX11 node protein) protein) in a random mixture by Vandoornft electrophoresis. Separate from the oligomer, remove the complex oligonucleotide by PCR, and cloned and then sequenced by double-stranded minibrenobu DNA sequencing. .

The present invention uses a binding site selection method that relies on the availability of PCH. in this way , the selected and amplified binding sites (SaABs) are characterized by protein binding characteristics. provide a unique sign. In a preferred embodiment, the method relies on consensus sequences. be done.

R Tsukikage and L Jo The present invention relates to oligonucleotides that specifically bind to proteins or other targets. Concerning methods for determining sequences. This method is particularly useful when the consensus sequence portion is known. is suitable for the DIIIA sequence. In this case, the protein being bound is or other targets is not necessarily required. This method is useful for certain The basic helinox-loop-helinox (bFfLH) protein, which is important for quality, particularly MyoD, cMYC and previously undescribed reticulocyte-derived applied to indicate nucleotide sequences that bind to different proteins.

Accordingly, in one aspect, the invention provides oligonucleotides that specifically bind to a target ligand. METHODS FOR DETERMINING CREOTIDE SEQUENCES. This method forms a random sector array. and a portion that allows for amplification of the oligomer. providing a mixture containing a target substance; treating the oligomer mixture with a target substance to target the oligomer mixture; forming a complex with an oligonucleotide that is specifically bound to it; conjugation separating the oligonucleotides from the unbound portion of the oligonucleotide mixture; a step of recovering the complexed oligonucleotides; and amplifying them. process; This method generally involves complexing, separation, and amplification. The round is repeated several times. Once a mixture with sufficient binding affinity is obtained, The sequence of recovered and amplified oligonucleotides complexed with the target is then make a decision In a preferred embodiment, oligonucleotides with random sequences The mixture further has a consensus sequence known to bind the target.

In other aspects, the invention provides oligonucleotides identified by the methods described above; and MyoDScMYc and reticulocyte-derived (7) It relates to oligonucleotide sequences that bind differently. In yet another aspect, the present invention is a target substance and a specifically bound oligomer in an environment free from cells. Concerning a complex containing.

In yet another aspect, the present invention provides an oligonucleotide having a sequence that specifically binds to a target substance. Sesame and the use of its oligomers in therapeutic, diagnostic, and purification processes. related.

[Lyu1rinya quantity plate FIG. 1 is a schematic illustration of the method of the invention.

Figure 2 shows the DNA arrangement of four oligomers used to illustrate the method of the invention. Indicates a column.

Figure 3 shows free oligonucleotides and bound MyoD-containing fusion proteins. Typical electrophoretic mobility shift (EMSA) of oligonucleotides This shows the separation results.

Figure 4 shows control and conjugated oligonucleotides recovered from the gel in Figure 3. Typical sequencing results obtained from the test are shown.

Figure 5 shows random and non-random oligonucleotide probes in vitro. Electrophoresis of complexes formed with proteins obtained by lobe transcription/translation. Dynamic Mobility Separation (EMSA) is shown.

Figure 6 shows comparative exposures of EMSA obtained from other conjugation reactions and the EMSA of Figure 5. The resulting higher exposure is shown.

FIG. 7 shows the results of the present invention after further rounds of complexation, separation, amplification and recovery. The results of separation of f17 sesame 10epsilon MSA recovered by the method are shown.

Figure 8 shows the control oligonucleotide mixture and the complex shown in Figure 7. Sequencing results of various oligomers selected from the resulting mixture are shown.

Figure 9 is a summary of oligonucleotide sequences obtained by the selection method of the present invention. Ru.

Figure 10 shows selected oligomers and complexes by binding to crude reticulocyte lysates. EMSA separation of proteins from myoblast and MEL cell extracts show.

Figure 11 shows that random EMSA results of recovered materials after selection from oligomers are shown.

Figure 12 shows the sequencing results of the recovered oligomers of Figure 11 after three rounds of selection. show.

& Maru 11 Joko j1” The present invention describes the use of oligomer sequences that specifically bind to desired targets, including proteins. Concerning methods by which collection and estimation can be made. Therefore, as a result of applying this method, Oligonucleotides with specific binding sequences can be prepared and It can be used in otide-based therapy and other applications.

For example, these oligonucleotides can be used to recover the substances to which they specifically bind. can be used as a separation means for Oligonucleotides with specific binding sequences For example, by force-pulling the otide onto a solid support, they are bound together. proteins or other cellular components can be recovered in useful amounts. Furthermore, this These oligonucleotides can be used in specific binding assays for target substances. It can be used for diagnosis by using Detectable moieties such as radioisotopes Once properly labeled using minutes, oligonucleotides that specifically bind Additionally, it can be used for in vivo imaging or histological analysis.

“Oligomer” or “oligonucleotide” can be used in single-stranded or double-stranded form. more than one RNA or DNA sequence of any of the following configurations, particularly one Contains short sequences such as dimers and trimers in either stranded or double-stranded form. be caught.

These sequences serve as intermediates in the production of specifically binding oligonucleotides. It can be a thing.

As used herein, "specifically binding oligonucleotide" , in an environment where other substances under the same environment are not complexed to this oligomer, An oligonucleotide that can form a complex with a desired target substance. general A minimum of about 10 nucleotides, preferably 15 nucleotides, is required for effective specificity. Required for physical binding. Regarding the binding specificity of the target/orthogonucleotide pair of the present invention The only obvious limitations are that there is sufficient sequence in the bound nucleotides to and the sufficient binding capacity of the target substance to obtain the desired interaction. target is placed If appropriate interactions are obtained under the conditions of the environment in which the Ligonucleotides may also be suitable. Therefore, there is almost no interference from other substances. the less specificity and strength of binding is required.

As further explained below, specifically binding oligonucleotides are Although it is necessary to have a sequence that confers a may be derivatized.

By applying the method of the invention, one or more oligonucleotides that specifically bind to a target After the oligonucleotides are identified, the specifically binding oligonucleotides are sequenced. , then resynthesized into any convenient form for the intended use. identified An oligonucleotide having a sequence or a deliberately modified form thereof can be used for this purpose. Since the original information identified by the method of the present invention can be newly synthesized based on the information of Gonucleotides can, in effect, have desired properties, such as increased permeability or nucleic acids. Both the backbone and substituted bases on that backbone may confer increased stability with respect to may contain variants for both. Therefore, in general, the results of the method of the invention The information obtained by analysis of the oligonucleotide boule obtained as Used in the synthesis of oligonucleotides with modifications.

In this way, an oligonucleotide having a sequence that specifically binds to a target substance is It may be a conventional DNA or RNA portion, or it may be a conventional DNA or RNA portion, or It can be a “modified” oligomer that has been The oligomers of the present invention are suitable for synthesis. Since it has been shown that it also contains intermediate products in the Any of the sil groups can be protected by standard protecting groups or activated. can be used for further linkage to other nucleotides, or can be attached to a solid support. It can be attached to a carrier. The OH at the 5° or 3° end is activated in the conventional manner. . The other 3° or 5° terminal OH may be protected. oligonucleotide product and intermediate products in which one or more phosphodiester linkages are replaced by other linking groups. It can be done. Such alternative linking groups include, but are not limited to, conventional linking groups. P(0)O of sulfodiester is P(0)S, P(0)NR2, P(0)RSP (S)S.

Embodiments are included in which P(0)OR', Co or CNR2 is substituted.

Here, R is H or alkyl (1-6C), and R is alkyl (1-6C). -6C), and this group also connects to the adjacent nucleotide via O or S. Can be combined. It is not necessary that all linkages in the same oligomer be the same.

The randomized portion of the oligonucleotide shown below originally consists of conventional bases, Contains adenine, guanine, totosine and thymine or uridine, but does not contain protein. Oligonucleotides incorporating similar forms of phosphorus and pyrimidine are also contemplated by the present invention. Included within the range.

"Analog" forms of purines and pyrimidines are known in the art and many are available. are used as chemotherapeutic agents.

Although it is impossible to list them exhaustively, the following are examples: 4-acetylintone, 5-(carpoquine hydroxylmethyl)uracil, 5- Fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl- 2-thiouracil, 5-carboxymethylaminomethyluranol, dihydroura Sil, Inonne, N6-Itsupentenyl adenine, 1-methyladenine, 1- Methyloneudouracil, 1-methylguanine, 1-methylinonne, 2.2- Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcyto syn, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- Methylaminomethyluracil, 5-methoxyami/methyl-2-thiouracil, β-D-mannone queosine, 5° methoxycarbonyl Methyluracil, 5-methchinuracil, 2-methylthio-N6-benteny Luadenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxy Acetic acid (V), wybutoxosine, pseudouraci , pheosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thio Uracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxy Acetic acid methyl ester, uranium-5-onaneacetic acid (V), pseudouracil, fluoride eosin, 2-thiocytosine, and 2,6-diamitpurine.

Most simply, conventional bases are used to apply the method of the invention. Pudding and substitution of pyrimidines with analogous forms may be advantageous in the design of final products. .

Oligonucleotides containing specific binding sequences identified by the methods of the invention are They can also be derivatized in a variety of ways.

For example, oligonucleotides with specific binding sequences can be used to separate target substances. When used in a conventional manner to allow chromatographic separation derivatized onto a solid support. Oligonucleotides label cellular components or when used to bind a detectable moiety to a target. , oligonucleotides can be derivatized to contain radioactive nuclei, fluorescent molecules, chromophores, etc. It will be done. If the oligonucleotide is used for specific binding assays, a solid support is It is desirable to bind it to a carrier or a detectable label. used for treatment Ligands that readily cross the cell barrier and promote therapeutic efficacy have a toxic moiety or an enzymatic activity that performs the desired function in the targeted region. It can be derivatized as follows. Additionally, the oligonucleotide can be contained in an appropriate expression system. may provide for in 5 in situ production of the desired sequence.

Generally, the methods of the present invention identify and, if necessary, native or The newly synthesized oligonucleotide, either in modified form, can be used as an antibody or its are useful in a similar manner to specific immunoreactive fragments of. Oligos of the invention Nucleotides bind specifically to target molecules of interest in both single and complex environments. characterized by the ability to match Therefore, the oligonucleotide-target complex Formation can be formed by methods similar to those used in immunoassay methods. child A wide range of protocols are known in the art, such as direct and competitive Extensive use of detection methods is also encompassed in the protocol, including both different forms of detection.

Similarly, antibodies have found diagnostic and therapeutic applications, as well as the control of cell proliferation and differentiation. As can be used, the oligonucleotides of the invention can also be used.

Oligonucleotide stone by day used as starting material in the method of the invention for determining specific binding sequences Oligonucleotides can be single-stranded or double-stranded DNA or RNA. Ru. Double-stranded DNA is preferred.

In any case, the starting material oligonucleotide is the oligonucleotide recovered from the complex. flanked on both sides by primer sequences that can accommodate replication chain reactions to oligonucleotides contains a randomized sequence portion. These flanking sequences are further amplified. may contain other convenient forms such as restriction sites to allow cloning of sequences .

The randomizing part uses a mixture of nucleotides at the positions you want to randomize, Can be constructed by conventional solid phase methods. The hot topic, any degree of randomization can be used. Some positions have only two or three positions instead of the traditional four. Can be randomized by a mixture of bases. The randomization position is the specified position can be changed by In fact, some candidate randomized sequence parts are actually known If so, it helps. Target sequences, as illustrated in the Examples below. is a protein for which the consensus sequence is known.

The method of the invention is exemplified by using proteins as target substances. but large enough to be specifically recognized by the oligonucleotide sequence. Any ligand can be used as a target. Therefore, glycoproteins, proteins proteins, carbohydrates, membrane components, receptors, lipids, organelles, etc. as complex targets. can be used. However, as exemplified below, this method It is very helpful if the census sequence is known. A particular example of this adaptation is The following examples include the characterization of a number of proteins involved in tissue growth and differentiation. The basic HL) I domain has been described. These proteins are A helix thought to mediate protein multimerization - to the lubou The plex region includes a region of basic amino acids followed in the sequence (N-C). Ma Multimerization positions the basic region to specifically bind DNA.

The DNA sequence that binds to the protein containing the bl(Ll) region is a palindromic It is already known that CANNTG has a system consensus region CANNTG. bHLH The protein containing region is gene E2A.

MyaD (related to myogenesis and muscle-specific gene expression), cMYc ( oncogenes), and growth-related pond genes listed below. be born. Presence of this Funcensus sequence and availability of the corresponding protein assists in the application of this method. However, this method is effective even if the target is available. For example, it can be applied even without consensus. This method also Adapted for recovering proteins, especially those with consensus sequences or known sequences. It can be done.

An overview of the method of the invention is shown in FIG. This method step allows for the selection and amplification of The binding site J (SaABs) is obtained.

As shown, the mixture of oligonucleotides is placed in the intended binding site. Synthesized with random arrangement.

This random sequence hybridizes to the primers used for PCR. bordered on both sides by appropriate areas.

As shown in item 1 of Figure 1, single-stranded DNA consists of random nucleotide sequences NNN It is prepared with Here, region A for primer hybridization is shown at the 3' end. Oligomers are synthesized into corresponding strands. Formed into double strands. The oligomer is thus primed by primer hybridization. tion areas A and B. This is targeted, in this case, along with the protein. Incubate the complex shown in item 3 by electrophoretic migration a (EMSA). separated from uncomplexed duplexes. The bound template is cleaved by PCH. amplified for sequencing. The double-stranded oligonucleotide under item 2 is also and amplified as a control. The resulting amplified sequence was run on a sequencing gel and selected. The nature of the binding partner of the selected random nucleotide rABCj is determined. times Using the recovered and amplified duplex, repeat this entire process until satisfactory results are obtained. Go back.

The method shown in FIG. 1 is merely illustrative. The mixture of oligonucleotides is Consists of single-stranded DNA or RNA, as well as double-stranded DNA as shown. obtain. At this stage, the primer sequences are distributed on single-stranded oligonucleotide strands. Adjacent to both sides of the dam section. Separation of the part of the mixture that binds to the target substance can be achieved by any It can be done in any convenient manner. For example, with unbound oligonucleotides Rather than relying on differences in electrophoretic mobilities of compared complexes, it is possible to can be attached to a support and the oligonucleotide mixture can be applied to this support. . The mixture of moieties that could not bind to the bound target is only washed from the support; Leaving behind the complexed portion of the mixture. Therefore, in general, this method Simply complexing a mixture with a target, or complexing something that could not be complexed. separation of oligonucleotides and complexed oligonucleotides by amplification. This includes cutting out the code. Amplification of oligonucleotides that bind to the target is a complex Either while still native or by pre-complexing oligonucleotides from the target. This can be done after the separation.

Generally, one or more "rounds" of binding, separation, and amplification of complexes are performed as appropriate. is required to obtain one set of oligonucleotides that bind to. this process is the subset of recovered bound nucleotides as starting material for the next round Simply repeat until a mixture with sufficient binding affinity is obtained using It will be done. Generally, the consensus sequence in the oligomer that binds specifically is determined. Therefore, it would be desirable to sequence this specifically binding subset. mentioned above Members of this subset can then be synthesized de novo, such that . thus, Oligonucleotides containing base modifications, backbone modifications, or both It can be prepared.

1 of 1 Therefore, the oligomers of the present invention having specifically binding nucleotide sequences Useful in clinical, diagnostic and research contexts.

For therapeutic indications, oligomers are generally used as oligonucleotides, as described above. Oligonucleotides, such as those used herein, are utilized in a manner suitable for therapeutic treatment. Otide therapy includes any use of oligonucleotides as pharmaceuticals. child This involves targeting specific NA or RNA, or complementation. or any other specific binding means, e.g. sequences in the major groove of a DNA double helix. Target any substance in the pond via specific orientation, or any other specific binding mode. It includes any of the following: For such treatments, the oligomers of the present invention may be used. The drug is formulated for various modes of administration, including systemic and local administration, or localized administration. can be avoided. The methods and formulations are generally based on Remington's Phar maceutica 25 sciences, Mack Publishing Co., Easton, PA, latest edition.

For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injection. stomach. For injection, the oligomers of the invention are in solution, preferably in Hang's solution. or formulated in a physiologically suitable buffer such as Ringer's solution. In addition, oligo The mer can be formulated in solid form and redissolved or suspended immediately prior to use. frozen Dried forms are also included.

Additionally, systemic administration may be by transmucosal or transdermal means; The compound may be administered orally. For transmucosal or transdermal administration, permeable A permeate suitable for the barrier is used in the formulation. Such permeate is generally For example, for transmucosal use, bile salts and fundic acid derivatives are known. be caught. Additionally, surfactants can be used to promote permeation. Transmucosal administration For example, by nasal spray or by the use of suppositories. For oral administration , oligomers are available in traditional oral dosage forms such as capsules, tablets, and tonics. Prescribed.

For topical administration, the oligomers of the invention may be formulated into ointments, salves, salves, etc., as known in the art. It is formulated as a gel or cream.

Oligonucleotides can also be used in expression systems, e.g. for gene therapy applications. Administered according to a method suitable for

In addition to therapeutic uses, the oligomers of the invention are useful for targeting targets to which they specifically bind. It can be used as a diagnostic reagent to detect the presence or absence of a substance. This way Such diagnostic tests involve contacting a sample with oligonucleotides that specifically bind. This is done by obtaining conjugation and then detecting this complex by conventional means. example For example, oligomers can be labeled using radioactive, fluorescent, or chemical labels. The labeling substance can be immobilized through specific or non-specific binding means. The presence of a label bound to the body support is detected. Alternatively, specific binding oligomers can be used to effect the initial conjugation to the support. specific binding A single stage for performing assays using such oligomers as a carrier. are commonly known for standard specific binding partner-based assays. .

The specifically binding oligomer of the present invention interferes with or inhibits the activity of the target substance It is suggested that the mechanism is not always established and is not part of the invention. can be done. The oligomers of the present invention target the mechanism or the mechanism of its effect. It is characterized by its ability to target specific substances, regardless of its origin.

To be used in research, the specifically binding oligonucleotides of the invention must be This makes isolation and purification of substances to which they bind particularly effective. For this application, typical Specifically, an oligonucleotide with a specific binding sequence is attached to a solid support. , used as an affinity ligand in the chromatographic separation of target substances be done. Affinity ligands further enhance the binding between the intended target and the unknown protein. Due to similarity, previously unknown substances can be extracted from material sources that do not contain the target substance. can be used for recovery. In addition, non-oligonucleotide/oligonucleotide As more data are accumulated on the nature of tide-specific binding, the effects of gene expression on Insights into the mechanisms of control can be made.

The following examples are meant to be illustrative and are not meant to limit the invention. stomach.

(Margin below) Actual JLf increase DNA is 4■oD Totanbaku Synthesize oligonucleotide sequences in randomized mixtures using standard solid-phase synthesis methods 2, which is shown in FIG.

As shown in Figure 2, MCK (muscle creatine kinase enhancer) It is a natural sequence known to bind to oD. Oligomer D1, D2 and D3 have various sequence randomized configurations, and additionally 5° and 3° Contains regions for binding to PCR primers shown as terminal B and Ao. Ru. Primer A is 5°-TCCGAATTCCTACAG-3' and primer B is , 5'-AGACGGATCCATTGCA-3°.

These conveniently have restriction enzyme sites. The double-stranded DI-D3 template was Annealing the oligonucleotides to a 10-fold molar excess of A, E, co li Synthesize complementary strands using the Flenow fragment of DNA polymerase, Generated by purifying the template on a 2% polyacrylamide gel. Its casting Kinase reaction of 733 type, Davis, R.L., et al., Kyoyu (1990) The ends were labeled with The MCK double-stranded template was annealed to its complement, cinchona. obtained from enzyme-treated oligonucleotides.

As shown in Figure 2, in Dl and D2, the randomization is and delete the consensus sequence part. In D3, randomization is performed by upstream Two nucleotides, two downstream nucleotides, and a consensus motif is limited to two nucleotides between members of

The conjugation was performed using approximately 200 ng of glutathione-MyoD bacterially produced fusion protein; and 0.15 ng of MCK template or 0.30 nH of random sequence template ( Approximately 6 x 10' cpll each) La5sar, except that 1100n poly(di-dC) was used for the It was carried out as described in A, B, et al., Ce11 (1989) 58:823. E MSA on a 6% polyacrylamide gel, Davis.

It was carried out according to the description of R, L, et al., Germany (1990) Teinia 33.

Glu-MyoD was incubated with MCK, Di, and D2. The results of MSA are shown in FIG. Randomized mixture as shown in the figure Because many of the oligomers in the MC have inappropriate sequences, the fusion protein It binds easily to sequences and less to Dl and D2.

To re-isolate the complexed template, 3 MM (Whatman) vapor bar was used. Sections approximately 0.3 cm wide were cut from the dried gel containing the packing. That gel cut The pieces were soaked in 0.5 ml of 0.5 M ammonium acetate, 10 mM at 37°C overnight. Incubated in gCl, 1 mM EDTA, and 0.1X SDS . Approximately 50% radioactivity was recovered. After adding 5 μg of tRNA carrier, The extract was treated with phenol and chloroform:isoamyl alcohol 241, respectively. Extracted twice and precipitated with ethanol. Place the precipitate in 0.3M sodium acetate, Then, it was precipitated again with ethanol.

Approximately 175 of the resuspended samples were purified using primers A and B, 5aiki, R , in PCR Technolo, A.J., Ehrlichlii (S. Tockton Press, NY) 1989. Listed in pp. 7-16 The 100 μm reaction was amplified by 35 cycles of PCR under standard conditions. The Mg"' concentration was then optimized. By carefully controlling the conditions, 11) g of the starting template Approximately 100 ng of product was obtained in the test reaction containing . Cut out from EMSA 30-100 ng of DNA was obtained in the reaction performed with the same material. The reaction product, 14 % polyacrylamide gel, eluted and purified as previously described.

Next, the recovered and amplified complex oligomers were combined with labeled Bly7-A or B and United 5tates Biochemic 31 ( :0. Sequencing was performed by the termination step of the commercially available enzyme enzyme method as follows. Ta. Primers were labeled at 1-2×10’ cpm/ng by kinase reaction. Recognize and remove unincorporated labels using a 5ephadex GSO spin column did. Mix 10 ng of labeled primer with approximately 5 ng of purified oligomer and add 1 μl of Sequenase Mn” buffer and 2 μl of 5x Sequenase buffer. Sequencing was performed on 12 μl reactions. Incubate reactions at 95°C for 5 minutes and then quickly centrifuged for 1 minute at room temperature.

The reaction was placed on water and added with 1 μl of 0.1 M dithiothreitol and Dilute/add 2 μm of Quenase 2.0 enzyme (1.8 in ice-cold TE, pH 74). I got it. Add 35 μm of this mixture to 25 μm each of the N-Quenase dGTP termination mixture. and incubated at 45°C for 4 minutes. Stop reaction/-quenase 4 μl of solution (reactions performed with dlTP termination mix minus Mn“2 buffer) It was concluded by adding.

The reaction was transferred to a 14% denatured polyacrylamide sequencing gel containing 8M urea in TBE. I put it on ru. Non-random bases appearing in C lane compared to G, A and T lanes excludes the "C" reactant of the sequence generated by primer B as it is generally thin , 1.5 μm of reaction was placed in each well. This difference is equivalent to 2.5 μl of C reaction. It was compensated by multiplying. Gels were fixed in 10% acetic acid and 10% methanol. Before treatment, a large excess of unreacted primer was removed to prevent its diffusion.

The results of sequencing the bands in FIG. 3 are shown in FIG. 4. As shown in Figure 4, the consensus Preferential recovery of consensus sequences was obtained from the randomized portion of the sequences. Sara In addition, at position 4 (see Figure 2) there is a cytosine different from the one present in the MCK sequence. Therefore, thymidine was preferentially present.

i taneoka l DNA 1 that makes each country's music 1. By the method of Example 1, the important consensus sequence for binding to MyoD was confirmed. The D3 oligomer with this sequence was used in the next study. D3 is Has a consensus sequence or is randomized in close proximity. MyoD, E Various tanonocrine proteins associated with differentiation, including 2A, E12 and E47, It was synthesized by in vitro translation by N.A. Some of them are Murre.

Reported in C9 et al., Cell (1989) 56:777. transcription sequence , mouse MyoD cDNA, human E12 cDNA (E12R) and human E 47 cDNA) et al., Benezra, R. et al. (1990): 49. Next, 50 μl reticulocyte lysate (Promeg Approximately 2.5 μl of the in vitro translation reaction of a) was combined with the randomized oligomer. used to test. homodimer, homomultimer, heterodimer , and heteromultimers were formed from these protein products. Heteroma To form multimers, the separate translation reactions are mixed before the DNA joins, The binding reaction was incubated for 20 minutes at 37°C before being added to the cocktail. Tan The protein preparation was incubated with either D2 or D3 as follows. I did it.

The final binding reaction for testing randomized oligomers was a 20 arm H epes, pH 7, 6, 50mM KCI, 1mM dithiothreitol, 1 mM EDTA, 8% glycerol, 0.1 μg poly dl/dC, and so Contains 2 μg of bp single-stranded oligonucleotide, both of which are free from non-specific competition. It was added as a mixture.

Each binding reaction contains approximately 6.9 x 10 in vitro synthesized protein species. M and 0.15 ng, providing a protein:DNA molar ratio of approximately 0.18. of MCK or 0.30 ng of D2 or D3 labeled template. join The reaction was run for 20 minutes at room temperature and EMSA was performed immediately.

The results of EMSA performed on these incubation mixtures are shown in Figure 5. . These results show that MCK is E12/MyoD, E47, and E47/MyoD. It shows that D3 binds strongly to oD and that D3 binds only to E47. deer and in addition to the gel for a similar reaction mixture using oligomer D2, more Long exposed gels (Figure 6) showed complexation of MCK with all of the test samples, and D 3 with E12/MyoD, and E47/MyoD in addition to E47. It shows a complex with.

For the bands cut out from the gel shown in Figure 6, incubation, EMSA and ['CR amplification was performed for three more rounds.

In such successive rounds, approximately 5 ng of purified amplified template was added to a standard PCR reaction. 30 μCi of 32P dTP, 50 am each of dATP in buffer.

PCR containing dGTP and dCTP and 100 ng each of A and B Labeling was performed in 20 μm reactions per cycle. React with a large excess of primer. was added to ensure that synthesis occurred on all templates. Signs not captured was removed using a 1 ml G50 spin column to confirm that the reaction product was full-length. I confirmed it. The binding reaction and EMSA were performed at a protein:DNA molar ratio of approximately 0.5. 4, except that about 0.1 ng of the PCR-labeled template pool was added. went like that.

Further conjugation was found as subsequent rounds were enriched with bound species. figure As shown in Figure 7, the compound predicted to confer sequence specificity compared to the control The fusion is between D3 and the target proteins MyoD, E12/MyoD, and E12/MyoD. 47 and E47/MyoD. D2 is E12/MyoD, complexed with E47 and E47/MyoD. Importantly, Figure 7 Furthermore, reticulocyte factor, rather than other target sequences, is also targeted by selective D3 oligomers, especially , E12, E12/MyoD and E47/MyoD selected oligomers. Indicates that they will be combined.

Figure 8 shows some of the complexes shown in Figure 7 cut out of the gel and arranged as described above. Shows the result of column determination.

Figure 8A shows the D3 control showing complete heterogeneity at six randomized positions. The results are shown for the mixture. DIIA sequences selected by the method of the present invention (Fig. 8B-8F) showed positional preference. For example, selection by complexing with MyoD The selected D3 oligomer (FIG. 8B) has obvious differences to T at positions 5 and 4. showing preference, positions 1 and -1 remain with some heterogeneity, positions -4 and -1 5 showed some preference for A. Reticulocyte lysate shown in Figure 8F clearly recognizes the sequence (G/A)CCAGTTG(N)A.

A summary of the binding and sequencing experiments shown in FIG. 8 is shown in FIG.

Preset position selection is indicated by shading and indicates absolute or near-absolute assigned preference. Precedence is indicated by uppercase letters, and incomplete preference is indicated by lowercase letters. However, the characters The bar above indicates the exact opposite; the base is in the indicated position (uppercase). Never found or only weakly presented (lower case).

Mitate Eyu D3 punishment for speaking P2 myoblasts (Lassar, A, B, et al. (1986) 47:649 ) and murine erythroleukemia (MEL) nuclear cell extracts were combined as described above, E It was used as target protein source for MSA and amplification rounds. P2 myoblast extraction Dignam, J.D., et al., Nu., except that the extract was not dialyzed. As described in Cleic Ac1ds Res (1983) 11:1475. and prepared it. MEL cell extracts were extracted from Gorski, K et al., Cell (198 6>47.767. D3 from MCK and lysate Both templates were used in conjugation reactions performed as described above under the following conditions: . P2 myoblast binding reaction product was 20mM Hepes, pH 7,6,1,5 mM MgCl2.50mM NaCl 5% glycerol and 500n Contains g-poly(di/dC). MEL cell binding reactions were carried out using 2 μg of ports for each reactant. The method described in Example 1 was followed except that Lidi/dC was included. both The binding reaction was incubated for 20 minutes at room temperature and then immediately incubated with 5% polyacrylic. EMSA was performed on an amide gel at 200V and 4°C. The results are shown in Figure 10. . As shown, D3 selected from the lysate is a factor in the MEL extract. and to factors different from those bound by MCK in myoblast extracts. match. Therefore, these previously unidentified target proteins are It is recovered by its ability to bind to D3 selected from substances.

Mitatego I CMYC Nbaku ni NA1 Bacteria containing the C-terminal 92 amino acids of human cMYc (cMYc-C92) The produced glutathione s-transferase (GST) fusion protein is Used as protein. This fusion protein contains the bHLH domain and the Contains ninzinova. The DNA template used was the above-mentioned DNA template on both sides of the consensus sequence. 2 with a random sequence flanking the A and B primers. It was D6. Several rounds of abdominal coalescence, EMSA and amplification are shown in Figure 11. required to recover the indicated preferred DNA binding sequence. Lane 2 in Figure 11 and 3 are the results from the second and third rounds of complexation/separation/amplification cycles. Show results.

Figure 12 shows the sequencing results. Amplified D3 was used as a control. Ta. Two bases were identified within the consensus sequence as shown in the figure. However, heterogeneity remains in the adjacent sequences.

5'-CCCCCCCAACACCTGCTGCCTGA-3' MCK5[F] ACCCCCCAA[EEECTGCTGCCTGAT3" D151■AC CCCCC 匪幀正TG 匪GccrGAr＠31D35' TG plate■CTGAT℃31D65'-CCCCCCACCGTGGTGCCT GA-3' CMIFIG, 2 GIuMyoD FIG.3 GATCGATCGATCGATC l 2 34 5 6 78 9101112MCK D3 FIG.6 FIG.9 MCK meltable products D3 cMycD63 GATCGATC Copy and translation of amendment) Article 184-8 of the Patent Law submitted on March 19, 1993

Claims (13)

[Claims] 1. A method for identifying oligonucleotide sequences that specifically bind to target substances. hand, the target substance with a mixture of randomized oligonucleotide sequences; Incubation under conditions where complexation with some, but not all, members occurs. bate, process; Conjugate oligonucleotides from unconjugated oligonucleotides step of separating the complex; recovering the complexed oligonucleotide from the complex; and amplifying; and Optionally, determining the sequence of the recovered oligonucleotide; A method that encompasses. 2. the target substance is known to bind to the consensus sequence, and The oligonucleotide mixture is randomized at the consensus sequence position. 2. The method of claim 1, wherein the method is not. 3. 2. The oligonucleotide mixture according to claim 1, wherein the oligonucleotide mixture is double-stranded DNA. Method. 4. The target substance is a protein, and the target substance is a basic loop-helix-loop. (bHLH) region, and the consensus region is CANNTG. The method described in claim 3. 5. The separation is performed by electrophoretic mobility shift assay (EMSA) and Claim 1, wherein the recovery and amplification are performed by replication chain reaction (PCR). Method described. 6. In the recovery of oligonucleotide sequences that specifically bind to the target substance, A mixture of oligonucleotide segments useful as a substance, the mixture comprising: comprising a randomized set of nucleotide sequences, in each member of the set The segment with the randomized DNA sequence is used as a primer for PCR. – A mixture that is flanked on both sides by an array. 7. 9. The mixture of claim 8, wherein the segment is a double-stranded DNA segment. . 8. The claim has a sequence specific to the target substance and is in a purified and isolated form. An oligonucleotide identified by the method described in 1. 9. The oligonucleotide according to claim 8, wherein the target substance is MyoD or cMYC. Gonucleotide. 10. A method for recovering a target substance from a sample, the method comprising: contacting the sample with at least one oligonucleotide having a sequence that a step in which the oligonucleotide comprises the substance and the oligonucleotide; identified in the method of claim 1 under conditions that form a complex; separating the complex from other substances in the sample; and separating the substance from the complex. A method comprising: recovering. 11. A method of modifying a target cell or tissue of a subject, the method comprising: specific substances specific to the target cell or tissue. comprising administering an oligonucleotide having a sequence that binds to the The gonucleotide is optionally derivatized with a moiety that enhances the modification. and the oligonucleotide is identified by the method of claim 1. method. 12. A method for determining the presence or absence of an analyte in a sample The law is When an analyte is present, the sample has a sequence that specifically binds to the analyte. The oligonucleotide forms a complex between the oligonucleotide and the analyte. and detecting whether the complex is present or absent; encompasses, A method, wherein said oligonucleotide is identified by the method of claim 1. 13. Target substance and at least one specifically bound oligonucleotide 2. A complex having the same oligonucleotide as described in claim 1. A complex that does not contain cellular components.