JP2008206524A - Nucleic acid ligand and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identification and production of improved nucleic acid ligands based on the SELEX process and to provide nucleic acid ligands to the HIV-RT, HIV-1Rev, HIV-1tat, 5 thrombin and basic fibroblast growth factor proteins. <P>SOLUTION: The method for producing an improved nucleic acid ligand from a candidate mixture of nucleic acids, which nucleic acid ligand is a ligand of a given target comprises: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; (c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; (d) repeating steps (a)-(c), as necessary, to identify a nucleic acid ligand; (e) determining the nucleic acid residues of the nucleic acid ligand that are necessary for binding target; and (f) producing improved nucleic acid ligand based on determination. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

FIELD OF THE INVENTION Described herein are methods for identifying and manufacturing nucleic acid ligands. A nucleic acid ligand is a double-stranded or single-stranded DNA or RNA species that specifically binds to a target molecule of interest. This identification of nucleic acid ligands is based on a method called SELEX, which is an acronym for “Systematic Evolution of Ligands for Exponential enrichment”.

  The method of the present invention comprises analyzing and applying information obtained from the SELEX method to create improved nucleic acid ligands for selected targets. These methods include computer modeling, boundary measurement methods and chemical modification methods. According to the method of the invention, 1) which nucleic acid residues of a nucleic acid ligand are critical for binding to a selected target; 2) which nucleic acid residues affect the structure of the nucleic acid ligand; And 3) It is possible to determine what the three-dimensional structure of the nucleic acid ligand is. This information enables the identification and production of improved nucleic acid ligands with improved structural stability and superior target binding ability. This information can also be used to produce non-nucleic or hybrid nucleic acid species that also function as ligands to the target. The methods of the present invention further allow analysis of target species used in the preparation of therapeutic and / or diagnostic methods.

Specifically, in the present invention, high affinity nucleic acid ligands for HIV-RT, HIV-1 Rev, HIV-1 tat , thrombin, and basic fibroblast growth factor (bFGF) protein are described. Modified nucleic acid ligands and mimetic ligands characterized by the nucleic acid ligands identified in the present invention are also within the scope of the present invention. Furthermore, nucleic acid ligands that can modify the biological activity of the target molecule (eg, nucleic acid ligands that inhibit the action of bFGF) are also within the scope of the present invention. Still further, nucleic acid ligands containing modified nucleotides are also included in the present invention.

BACKGROUND OF THE INVENTION Many proteins or small molecules are not known to bind specifically to nucleic acids. Exceptional known proteins are regulatory proteins such as repressors, polymerases, activators, etc. that function in living cells to cause the transfer of genetic information encoded in nucleic acids to the cellular structure and replication of genetic material It is. Furthermore, small molecules such as GTP bind to several intron RNAs.

Living organisms have evolved by limiting the function of nucleic acids primarily to the role of providing information. The central dogma proposed by Crick, through a replication process that "reads" information in the template nucleic acid and produces a complementary nucleic acid, whether the nucleic acid (RNA or DNA) is in its original or expanded form, It is proposed that it can act as a template for the synthesis of other nucleic acids. All experimental examples of genetics and gene expression depend on these properties of nucleic acids: essentially because of the chemical concept of base pairing , and the replication process in its relatively error-free manner. Therefore, information on double-stranded nucleic acids has a lot of duplication.

  The 20 natural amino acids that are individual components of proteins have chemical differences and activities sufficient to provide a very wide range of binding and catalytic activities. However, nucleic acids are thought to have a narrower chemical potential compared to proteins, except that they have a genetic role in transmitting genetic information from virus to virus, from cell to cell, and from organism to organism. Nucleotides that are nucleic acid components in this respect must have only a paired surface that allows duplication of information in Watson-Crick base pairs. The nucleic acid component need not have chemical differences and activity sufficient for a wide range of binding or catalysis.

  However, the few nucleic acids found in nature are involved in binding to certain target molecules, and even a few catalysis examples have been reported. The range of this type of activity is narrow compared to proteins (more specifically antibodies). For example, if a nucleic acid is known to bind to a protein target with high affinity and specificity, the binding depends on the exact sequence of nucleotides consisting of DNA or RNA ligands. For example, short double-stranded DNA sequences are known to bind to target proteins that repress or activate both prokaryotic and eukaryotic transcription. Other short double stranded DNA sequences are known to bind to restriction endonucleases, protein targets that are selected with high affinity and high specificity. Other short DNA sequences act as centromers and telomeres on the chromosome, possibly by generating ligands for the binding of specific proteins involved in the mechanism of chromosome action. For example, double-stranded DNA has the well-known ability to bind into the corners and nicks of target proteins that function for DNA binding. Single-stranded DNA can also bind to a small number of proteins with high affinity and high specificity, but there are few such examples. From the known examples of double-stranded DNA-binding proteins, the binding interaction will be explained on the assumption that various protein motifs that project amino acid side chains in the large groove of B-type double-stranded DNA are involved. It has become possible to examine sequences that give specificity.

  Double-stranded DNA often acts as a ligand for certain proteins (eg, E. coli endonuclease RNase III). Examples of target proteins that bind to single-stranded RNA are known, but in these cases the single-stranded RNA often forms a complex three-dimensional shape that includes local regions of intramolecular double strands. Amino-acyl tRNA synthetases bind strongly to tRNA molecules with high specificity. A short region in the genome of an RNA virus binds to the viral coat protein with strong and high specificity. The short sequence of RNA binds to DNA polymerase encoded by bacteriophage T4, which also has high affinity and high specificity. That is, it is possible to find double-stranded or single-stranded RNA and DNA ligands that are binding partners for specific protein targets. Many known DNA binding proteins specifically bind to double stranded DNA, while many RNA binding proteins recognize single stranded RNA. This statistical bias in the literature reliably reflects the current statistical trends in the biosphere, using DNA as a double-stranded genome and RNA as a single-stranded material that goes beyond its genomic role. ing. Chemically, there is no great reason to ignore single-stranded DNA as a sufficiently competent partner for specific protein interactions.

RNA and DNA were also found to bind to smaller target molecules. Double-stranded DNA binds to various antibiotics such as actinomycin D. Specific single-stranded RNA binds to the antibiotic thiostreptone; the specific RNA sequence and structure probably binds to other antibiotics (especially those whose functions inactivate the target organism's ribosome) To do. The family of evolutionarily related RNAs binds specifically to one of the 20 amino acids 5 (Jarus, M. (Yarus, M.) (1988) Science 240 : 1751) as well as specific and appropriate affinities. It binds to nucleotides and nucleosides with sex (Bass, B. (Bass, B.) and Seck, T. (Cech, T.) (1984) Nature 308 : 820). Catalytic RNA is also known, but the range of these chemical possibilities is narrow, and it has so far been mainly involved in phosphide transesterification and hydrolysis of nucleic acids.

  Despite these known examples, the majority of major proteins and other cellular components are believed not to bind to nucleic acids under physiological conditions, and such binding is nonspecific even if observed. It is believed that. The ability of nucleic acids to bind to other compounds is limited to the relatively few examples described above, or the chemical ability of nucleic acids for specific binding is avoided in the natural structure (selected against Have been). The present inventor said that nucleic acids can form virtually unlimited array shapes, sizes and configurations as chemical compounds and have a much wider range of binding and catalysis than shown in biological systems. These fundamental ideas are assumed.

In some known examples of protein-nucleic acid binding, chemical interactions have been studied. For example, the size and sequence of the RNA site for bacteriophage R17 coat protein binding has been identified by Uhlenbeck and co-workers. The minimal natural RNA binding site (21 base length) of the R17 coat protein is determined by the nitrocellulose filter binding assay, in which a variable-size labeled fragment of mRNA remains after the protein-RNA fragment complex binds to the filter. (Carey et al. (1983) Biochemistry 22 : 2601). To measure the contribution of individual nucleic acids to protein binding, many sequence variants of the minimal R17 coat protein binding site were generated in vitro (Uhlenbeck et al. (1983) J. Biomol. Structure Dynamics 1 : 539, and Romaniuk et al. (1987) Biochemistry 26 : 1563). Maintaining a hairpin loop structure at the binding site is essential for protein binding, but in addition, nucleotide substitutions of many single-stranded residues at the binding site can significantly affect binding, including the raised nucleotides on the hairpin axis. Was found. A similar study examined the binding of bacteriophage Qβ coat protein to translation operators (Witherell and Uhlenbeck (1989) Biochemistry 28:71). The Qβ coat protein RNA binding site is similar to that of R17 in size and predicted secondary structure in that it consists of approximately 20 bases with an 8 base pair hairpin structure containing raised nucleotides and a 3 base loop. It was found that. Unlike the R17 coat protein binding site, only one of the single-stranded residues of the loop is essential for binding and the presence of raised nucleotides is not necessary. This protein-RNA binding interaction associated with translational control exhibits a fairly high specificity.

Nucleic acids are known to form secondary and tertiary structures in solution. Double-stranded DNA includes the so-called B double helix type, Z-DNA and super helical twist structure (Rich, A. (Rich, A.) et al. (1984) Ann. Rev. Biochem. 53 : 791). . Single-stranded RNA forms a localized region of secondary structure, such as a hairpin loop or pseudoknot structure (Shimmel, P. (1989) Cell 58 : 9). However, little is known about the stability of the loop structure of unpaired loop nucleotides, the kinetics of formation and denaturation, the effect on thermodynamics, and the tertiary structure and three-dimensional shape, as well as the 3 Little is known about the kinetics and thermodynamics of tertiary folding (Tuerk et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 1364).

A type of in vitro evolution in RNA bacteriophage Qβ replication has been reported. Mills et al. (1967) Proc. Natl. Acad. Sci. USA 58 : 217; Levinsohn & Spiegleman (1968) Proc. Natl. Acad. Sci. USA 60 : 866; Levin Thorne and Spiegleman (1969) Proc. Natl. Acad. Sci. USA 63 : 805; Saffhill et al. (1970) J. MoI. Mol. Biol. 51 : 531; Kacian et al. (1972) Proc. Natl. Acad. Sci. USA 69 : 3038; Mills et al. (1973) Science 180 : 916. Phage RNA acts as a polycistronic messenger RNA that directs translation of phage-specific proteins and as a template for its own replication catalyzed by Qβ RNA replicase. This RNA replicase was shown to be highly specific for its RNA template. During the in vitro replication cycle, a small RNA variant that was also replicated by Qβ replicase was isolated. Small changes in the conditions for performing the replication cycle resulted in the accumulation of different RNAs, probably because the changed conditions were more suitable for their replication. In these experiments, the selected RNA was effectively bound by replicase to initiate replication and had to work with a kinetically suitable template during RNA elongation. Kramer et al. (1974) J. MoI. Mol. Biol. 89 : 719 reported the isolation of a mutant RNA template for Qβ replicase and reported that this replication was more resistant to inhibition by ethidium bromide than the native template. This mutant was not present in the initial RNA population but was suggested to be generated by sequential mutations during 20 in vitro replication cycles with Qβ replicase. The only source of mutation during selection was the intrinsic error rate during elongation by Qβ replicase. What is referred to as “selection” in these studies was initially caused by preferential amplification of one or more of a limited number of naturally occurring variants of uniform RNA sequences. There was no choice of the desired outcome, only what was inherent in the mode of action of Qβ replicase.

Joyce and Robertson (Joyce (1989) RNA: catalysis, splicing, evolution, RNA: Catalysis, Splicing, Evolution , Belfort and Shub (ed.), Elsevier, Elsevier Amsterdam, pages 83-87; and Robertson and Joyce (1990) Nature 344 : 467) reported methods for identifying RNAs that specifically cleave single-stranded DNA. The selection of catalytic activity was based on the ability of the ribozyme to catalyze the cleavage of the substrate ssRNA or DNA at a specific position and transfer the 3 ′ end of the substrate to the 3 ′ end of the ribozyme. By using oligodeoxynucleotide primers, the product of the desired reaction can only bind to the finished product via a linkage formed by a catalytic reaction, allowing selective reverse transcription of the ribozyme sequence. chosen. This selected catalytic sequence was amplified by ligating the T7 RNA polymerase promoter to the 3 ′ end of the cDNA followed by transcription of the RNA. This method was used to identify, from a small number of ribozyme mutants, mutants that are most reactive to cleavage of selected substrates.

  The prior art does not teach or suggest more than a limited range of chemical functions for nucleic acids in interactions with other substances (like binding to certain specific oligonucleotide sequences). As a target for evolved proteins; and more recently as a catalyst with a limited range of activities). Prior “selection” experiments have been limited to mutants with a narrow range of functions already described. Here it will be understood for the first time that nucleic acids have a very wide range of functions, and the methodology for realizing this possibility is disclosed herein.

  US patent application Ser. No. 07/536, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands for Exponential enrichment” by Gold and Turk. No. 428 and US Patent Application No. 07 / 714,131 (PCT / US91 / 04078), filed June 10, 1991, named “Nucleic Acids Ligands”, Gold and Turk. Also describes a radically new method for making nucleic acid ligands for any target of interest. All of these applications are collectively referred to herein as SELEX patent applications and are hereby incorporated by reference.

  The method of the SELEX patent application is that nucleic acids are capable of forming various two-dimensional and three-dimensional structures and ligands (specific binding pairs) with virtually any compound (large or small). Based on the unique insight of having sufficient chemical flexibility available within the monomer.

  This method involves the step-by-step iteration of selecting from a mixture of candidates and using the same general selection theme to achieve virtually any objective criteria for binding affinity and selectivity, and structural refinement. Including. A method referred to herein as SELEX starts with a mixture of nucleic acids (preferably consisting of sections of randomized sequences), contacts the mixture under conditions suitable for binding, and binds to the target molecule. Fractionating unbound nucleic acid from the nucleic acid, dissociating the nucleic acid-one target pair, amplifying the nucleic acid dissociated from the nucleic acid-one target pair to produce a nucleic acid mixture enriched in ligands, then binding, fractionating, Including a step of repeating the steps of dissociation and amplification as many times as necessary.

Without being bound by the theory of preparation, SELEX is based on the inventors' insight that there is a wide range of binding affinity for the target of interest in nucleic acid mixtures containing many possible sequences and structures. For example, a nucleic acid mixture consisting of randomized sections of 20 nucleotides has 4 ²⁰ candidate possibilities. Those with a high affinity constant for the target are most likely to bind. After fractionation, dissociation and amplification, a second nucleic acid mixture is generated and concentrated to obtain even higher binding affinity candidates. Gradually the best ligand is obtained by further selection until the resulting nucleic acid mixture consists primarily of only one or several sequences. They are then cloned, sequenced, and individually tested for binding affinity as a pure ligand.

The selection and amplification cycle is repeated until the goal is achieved. In the most common case, selection / amplification continues until no significant improvement in binding strength is seen with repeated cycles. This method can be used to collect as many as about 10 ¹⁸ different nucleic acid species. The nucleic acid of the test mixture preferably includes a randomized sequence portion with conserved sequences necessary for sufficient amplification. Nucleic acid sequence variants can be produced in a number of ways, including synthesis of randomized nucleic acid sequences and size selection from randomly cleaved cellular nucleic acids. The variable sequence portion may contain all or part of the random sequence; it may also contain a portion of the conserved sequence incorporated with the random sequence. Sequence changes in the test nucleic acid may be introduced or increased by mutagenesis before or during selection / amplification iterations.

  In one embodiment of the method of the SELEX patent application, the selection process is very effective in isolating the nucleic acid ligand that most strongly binds 5 to the selected target, with only one selection and amplification. It is only necessary. Such an effective selection is, for example, a chromatographic type in which the ability of the nucleic acid to associate with the target bound to the column functions to allow the column to separate and isolate the highest affinity nucleic acid ligand. Get up in the process.

  In many cases, it is not always desirable to perform a SELEX iteration until a single nucleic acid ligazod is identified. A target-specific nucleic acid ligand solution comprises a group of nucleic acid structures or motifs having many conserved sequences and many sequences that are replaced or added without significantly affecting the affinity of the nucleic acid ligand for the target. By terminating the SELEX process before completion, it is possible to determine the sequence of members of many nucleic acid ligand solution groups.

  It is known that primary, secondary and tertiary structures of various nucleic acids exist. The structures or motifs that have proven most commonly associated with non-Watson-Crick interactions are hairpin loops, symmetric and asymmetric ridges, false knots and myriad combinations thereof. Almost all known such motifs suggest that they are formed within a nucleic acid sequence of 30 nucleotides or less. For this reason, it is often preferred that a SELEX process dealing with adjacent randomized sections starts with a nucleic acid sequence containing random sections between about 20-50 nucleotides.

  The SELEX patent application also describes a method for obtaining a nucleic acid ligand that binds to a nucleic acid ligand that includes a nucleic acid ligand that binds to one or more sites of the target molecule and a non-nucleic acid species that binds to a specific site of the target. ing. This SELEX method provides a means to isolate and identify nucleic acid ligands that bind to any possible target. However, in a preferred embodiment, the SELEX method is applied in situations where the target is a protein (including both nucleic acid binding proteins and proteins that are not known to bind to nucleic acids as part of a biological function). The

  Little is known about RNA structure at high resolution. The basic A-type helical structure of double-stranded RNA is known from fiber diffraction studies. X-ray crystallography gave a few tRNAs and a short polyAU helix structure. The X-ray structure of the tRNA / synthetase RNA / protein complex has also been elucidated. One tetranucleotide hairpin loop and one model false knot have been revealed from NMR studies.

There are several reasons behind the lack of structural data. Until the emergence of in vitro RNA synthesis methods, it was difficult to isolate sufficient amounts of RNA for structural studies. Until the discovery of catalytic RNA, few RNA molecules were considered worthy of structural studies. It was difficult to obtain good quality tRNA crystals, and water was devoted to the study of other crystals. The technique of NMR studies of molecules of this size has only recently become available.

  As mentioned above, several catalytic RNA structures are known and SELEX technology has been developed to select RNAs that bind strongly to various target molecules, so new catalytic RNA structures can also be selected. It may be. In order to know exactly these actions and to use this knowledge to improve them, it has become important to know the structure of these molecules.

  It is preferable to fully understand RNA folding in order to be able to predict RNA structure with less effort than using exact NMR and exact X-ray crystallography. There has always been a demand for both proteins and RNA to be able to infer structure based on sequence and with limited (or no) experimental data.

  It is well known that the prediction of protein structure is difficult. First of all, the secondary and tertiary structures of proteins form cooperatively; protein folds are approximated thermodynamically by a two-state model in a fully folded state and a completely unfolded state. Can be done. This means that the number of degrees of freedom for modeling protein structure is very large; without predictable intermediates, the prediction problem cannot be broken down into smaller problems that can be handled smaller. In contrast, RNA often appears to produce a well-defined secondary structure that provides more stability than tertiary interactions. For example, the tertiary structure of a tRNA can be destroyed without destroying the secondary structure by chelation of magnesium or by increasing temperature. The prediction of RNA secondary structure is well understood, and for small RNA molecules it is generally very accurate. In RNA, structure prediction is broken down into sub-problems; first the prediction of secondary structure; the next is the prediction of how the resulting helix and the remaining single strands are arranged relative to each other.

  For RNA, the first attempt at structure prediction was made for tRNA. The secondary structure of the canonical tRNA clover leaf was revealed by comparative sequence analysis, and the problem was narrowed down to one problem of arranging four short A-type helices in space with each other. In order to create a tRNA model, there are some distance limitations derived from manual CPK modeling, minimization of easily calculated energy, and common cross-linking studies and phylogenetic variations. When used, and years later, when the initial crystal structure of phenylalanine tRNA was solved, it turned out to be unfortunately wrong.

  Computer modeling has replaced manual modeling, which has saved the difficulty of modelers posed by gravity and mass. Computer modeling can only be used without additional experimental data if, for example, homologous structures are known; for example, the 3 ′ end of the RNA genome of turnip yellow mosaic virus Was modeled based on the known 3D structure of tRNA and the knowledge that the 3 ′ end of TYMV is recognized as tRNA-like by many cellular tRNA modifying enzymes. This model is the first 3D model of an RNA false knot; the basic structure of the isolated model false knot has been confirmed by NMR data.

  In order to model the structure of several RNA molecules, chemical and enzymatic protection data were manually inspected and a computer modeling method was used. In one isolated substructure, NMR studies of isolated GNRA hairpin loops showed that one model of the GNRA four nucleotide loop form was essentially correct.

Francois Michel ((1989) Nature 342 : 391) constructed a model of the catalytically active core of the Group I intron. Like tRNA, the secondary structure of the Group I intron core is well known from comparative sequence analysis, so the problem is focused on one problem of correctly arranging the helix and the remaining single-stranded region. Michael (1989) visually analyzed a set of aligned 87 Group I intron sequences to detect common changes in triplets outside of seven strong pairs and secondary structures, which he It was interpreted as a third contact and manually incorporated into his model as a restriction. So far there is no independent confirmation of Michael's model.

  Other attempts have been made to devise automated methods to handle distance limitations from common changes in cross-linking, fluorescent transitions, or phylogeny. RNA is processed as a collection of cylinders (type A helix) and beads (single-stranded residues), and a mathematical technique called distance geometry creates an arrangement of these elements consistent with a set of distance constraints. Used to. Using a small set of seven distance restrictions on the tertiary structure of phenylalanine tRNA, this method created a common L-shaped tRNA structure about 2/3 times.

The HIV-1 tat protein activates transcription in the long terminal repeat (LTR) of the HIV-1 viral genome. See Cullen et al. (1989) Cell 58 : 423. The mechanism of activation is unclear or at least controversial, but the transcribed RNA needs to contain a unique hairpin structure (called TAR) with a trinucleotide ridge. FIG. 25 shows the site of interaction between natural TAR RNA and tat. The small basic domain of the tat protein has been shown to interact directly with the TAR RNA sequence. Weeks et al. (1990) Science 249 : 1281; Roy et al. (1990) Genes Dev. 4: 1365; Calnan et al. (1991a) Genes Dev. 5: 201. Arginine in this basic domain appears critical to the interaction. See Carnan et al. (1991a) supra; Subramanian et al. (1991) EMBO 10: 2311-2318; Carnan et al. (1991b) Science 252: 1167-1171. Arginine alone binds specifically to the TAR RNA sequence and appears to compete for tat protein binding. Tao et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2723-2726; Puglisi et al. (1992) Science 257: 76-80.

Tat- TAR interaction alone is insufficient to support transactivation; for co-binding of tat protein to TAR, and subsequent in vivo or in vitro transactivation, perhaps a cellular factor (68 kD loop binding protein )is required. Marciniak et al. (1990a) Proc. Natl. Acad. Sci. USA 87: 3624; Marcinique et al. (1990b) Cell 63: 791. Overexpression of TAR sequences in retrovirus-transformed cell lines makes them highly resistant to HIV-1 infection. See Sullanger et al. (1990) Cell 63: 601.

Thrombin is a multifunctional serine protease with important procoagulant and anticoagulant activities. As a procoagulant enzyme, thrombin clots fibrinogen, activates blood clotting factors V, VIII, and XIII and activates platelets. Specific cleavage of fibrinogen by thrombin initiates the polymerization of fibrin monomers, the first event of blood clot formation. A major event in platelet thrombus formation is platelet activation from “unbound” to “bound” mode, where thrombin is the most potent physiological activator of platelet aggregation (Berndt and Phillips (1981) Platelets in Biology and Pathology, JL Gordon (Ed.) (Amsterdam: Elsevier / North Holland Biomedical Press), 43- 74 pages; Hansen and Parker (Hansen and Harker) (1988) Proc.Natl.Acad.Sci.USA 85: 3184; Eight (Eidt) et al. (1989) J.Cl n.Invest 84:. 18). Thus, thrombin as a procoagulant plays a central role in bleeding prevention (physiological hemostasis) and vaso-occlusive thrombus (pathological thrombosis).

As an anticoagulant, thrombin binds to thrombomodulin (TM) expressed on the surface of vascular endothelial cells. TM changes substrate specificity from fibrinogen and platelets to protein C by a combination of allosteric changes in the arrangement of active sites and the overlap of TM and fibrinogen binding sites on thrombin. Activated protein C, Ca ²⁺ , and a second vitamin K-dependent protein cofactor present on the phospholipid surface, protein S, inhibits coagulation by factor Va and factor VIIIa that degrade the protein. Thus, the formation of thrombin-TM complexes converts thrombin from procoagulant enzymes to anticoagulant enzymes, and the normal balance between these opposing activities is critical to the control of hemostasis.

Thrombin is also involved in biological responses remote from the coagulation system (reviewed in Shuman (1986) Ann. NY Acad. Sci. 485 : 349; Marx (1992) Science. 256 : 1278). Thrombin is a monocyte chemotactic substance (Bar-Shavit et al. (1983) Science 220 : 728) and lymphocytes (Chen et al. (1976) Exp. Cell Res. 101 : 41). , A mesenchymal cell (Chen and Buchanan (1975) Proc. Natl. Acad. Sci. USA 72 : 131) and fibroblasts (Marks (1992) supra). Thrombin activates endothelial cells to express the neutrophil adhesion protein GMP-140 (PADGEM) (Hattori et al. (1989) J. Bio 1. Chem. 264 : 7768), producing platelet-derived growth factor. (Daniel et al. (1986) J. BioI. Chem. 261 : 9579). Recently, thrombin has been shown to cause neurite contraction by cultured neurons (reviewed by Marks (1992) supra).

The mechanism by which thrombin activates platelets and endothelial cells is through the functional thrombin receptor found in these cells. The putative thrombin cleavage site (LDR / S) of this receptor suggests that the thrombin receptor is activated by proteolytic cleavage of the receptor. This cleavage event “exposes” the N-terminal domain, which acts as a ligand and activates the receptor (View (Vu et al. (1991) Cell 64 : 1054).

  Vascular injury and thrombus formation are central events in the pathogenesis of various vascular diseases, including arteriosclerosis. The pathogenesis process of platelet and / or coagulation activation leading to various disease states and thrombi in various sites (coronary arteries, intracardiac and prosthetic heart valves) appears to be different. Thus, it may be necessary to use a platelet inhibitor, an anticoagulant, or a combination of both in combination with a thrombolytic agent to open the blocked vessel and prevent reocclusion.

  Controlled proteolysis by compounds in the coagulation cascade is critical for hemostasis. As a result, in 6 pathological conditions, where there are various complex control systems based in part on a series of highly specific protease inhibitors, functional production may result from excessive production of active proteases or inactivation of inhibitory activity. Inhibitory activity is disturbed. The persistence of inflammation in response to numerous trauma (tissue damage) or infection (sepsis) depends on both the plasma cascade system including thrombin and both lysosomal proteolytic enzymes. Multiple organ failure (MOF) in these cases is enhanced by an imbalance of concurrent proteases and their inhibitory regulators. An imbalance of thrombin activity in the brain causes neurodegenerative diseases.

  Thrombin is naturally inhibited during hemostasis by binding to antithrombin III (AT III) in a heparin-dependent reaction. Heparin exerts its effect by its ability to enhance the action of AT III. In the brain, the protease nexin (PN-1) would be a natural inhibitor of thrombin that controls neurite outgrowth.

  Heparin is a glycosaminoglycan composed of a chain in which D-glucosamine and uronic acid residues are alternately repeated. Currently, heparin is widely used as an anticoagulant in the treatment of unstable angina, pulmonary embolism, atherosclerosis, thrombosis, and subsequent myocardial infarction. Its anticoagulant effect is mediated by interaction with AT III. When heparin binds to AT III, the arrangement of AT III changes, resulting in a significantly enhanced thrombin inhibitor. Although heparin is generally thought to be effective for certain indications, the physical size of the AT III-heparin complex prevents access to many biologically active thrombins in the body and Reduces the ability to inhibit formation. Side effects of heparin include bleeding, thrombocytopenia, osteoporosis, skin necrosis, alopecia (alpe), hypersensitivity and hypoaldosteronism.

Hirudin is a medical leeches of Europe Hirudisu-Medishinarisu (Hirudis medicinalis) derived, it is a potent peptide inhibitor of thrombin. Hirudin inhibits all known functions of α-thrombin and has 101 additional sites (a high affinity site at or near the catalytic site for serine protease activity and a second anionic exosite). ) Kinetically binds to thrombin. Sites that are anionic outside also bind to fibrinogen, heparin, TM, and possibly receptors involved in mediating platelet and endothelial cell activation. The C-terminal hirudin peptide, which was shown to bind at an anionic outer site by co-crystallization with thrombin, probably fibrin formation, platelets and endothelial cells by competing for binding at this site. And has an inhibitory effect on protein C activation via TM binding. This peptide does not inhibit proteolytic activity against tripeptide chromogenic substrates, factor V or factor X.

  The structure of thrombin has become a particularly desirable target for nucleic acid binding due to its anionic external site. Site-directed mutagenesis at this site indicated that fibrinogen-clotting and TM binding activity were separable. Perhaps an RNA ligand with procoagulant and / or anticoagulant effects will be selected depending on how it interacts with thrombin (ie the substrate it mimics).

The single-stranded DNA ligand of thrombin was prepared by the same method as SELEX. See Bock et al. (1992) Nature 355 : 564. A consensus ligand was identified after performing a relatively low rotational SELEX, which proved to have the ability to prevent in vitro clot formation. This ligand is a 15 base DNA 5′GGTTGGGTTGGTTG-3 ′, referred to herein as G15D (SEQ ID NO: 1). The symmetry of the primary sequence suggests that G15D has a regular fixed tertiary structure. The kD of G15D to thrombin is about 2 × 10 ⁻⁷ . For effective thrombin inhibition as an anticoagulant, the stronger the affinity of the ligand for thrombin, the better. Basic fibroblast growth factor (bFGF) is a multifunctional effector for many cells derived from mesenchyme and neuroectoderm (Rifkin & Moscatelli (1989) J. Cell Biol. 109 ). 1; Baird & Bohlen (1991) Peptide Growth Factors and Their Receptors (Spawn, MB and Roberts, AB (Spawn, MB & Roberts, ed.). 369-418, Springer, New York; Basilico and Moscatelli (1992) Adv. Cancer Res. 59 : 115). This is the case of acidic FGF (Jay et al. (1986) Science 233 : 541; Abraham et al. (1986) Science 233 : 545), int-2 (Moore et al. (1986) EMBO J. 5 : 919), kFGF / hst / KS3 (Delli-Bovi et al. (1987) Cell 50 : 729; Taira et al. (1987) Proc. Natl. Acad. Sci. USA 84 : 2980), FGF- 5 (Zhan et al. (1988) Mol. Cell. Biol. 8 : 3487), FGF-6 (Marics et al. (1989) Oncogene 4 : 335) and keratinocyte growth factor / FGF-7 (Finch) (Finch) (1989) Science 245 : 752) and is one of the most studied and best characterized members of the family of related proteins.

In vitro, bFGF stimulates cell proliferation, migration, induction of plasminogen activator and collagenase activity (Presta et al. (1986) Mol. Cell. Biol. 6 : 4060; Moscateri et al. (1986) Proc. Natl.Acad.Sci.USA 83 : 2091; Mignati et al. (1989) J. CellBiol. 108 : 671). In vivo , this is one of the most powerful inducers of neovascularization. Its in vivo angiogenic activity is not only tissue repair and wound healing, but also disease states characterized by pathological neovascularization (eg tumor growth, tumor metastasis, diabetic retinopathy and rheumatoid arthritis) It also suggests a role in odor (Folkman & Klagsbrun (1987) Science 235 : 442; Gospodarowitz (1991) Cell Biology Reviews 25 : 307).

bFGF has no signal sequence for secretion but is probably carried by exocytosis and found on both sides of the plasma membrane (Vlodavsky et al. (1991) Trends Biol. Sci. 16 : 268; And Rifkin (1991) J. Ce11. Biochem. 47 : 201). In the extracellular matrix, this is typically associated with a fraction containing heparan sulfate proteoglycans. Indeed, heparin affinity chromatography is a useful method for the purification of this and other heparin-binding growth factors. bFGF binds to low and high affinity sites in cell culture. The low affinity site consists of cell-associated heparan sulfate proteoglycans to which bFGF binds with an affinity of about nanomolar (Moscateri (1987) J. Cell. Physiol. 131 : 123). All biological effects of bFGF are mediated by interaction with the high affinity binding site (10-100 pM), a dimeric tyrosine kinase FGF receptor (Ueno et al. (1992) J. Biol. Chem. 267 : 1470). To date, five FGF receptor genes have been identified, each of which can produce several structural variants as a result of alternative mRNA splicing (Armstrong et al. (1992) Cancer Res. 52: 2004; Ueno et al. (1992) supra). To date, there is considerable evidence that the low affinity binding site and the high affinity binding site act in concert to determine the overall affinity of bFGF. For mutant cell lines deficient in glycosaminoglycan synthesis (Yayon et al. (1991) Cell 64 : 841) or heparitinase treated cells (Rapraeger et al. (1991) Science 252 : 1705) Experiments demonstrated that binding of heparin sulfate added externally to bFGF requires a signal via a tyrosine kinase receptor in the absence or absence of cell-associated heparan sulfate. The recent results of degradation of the observed Kd into kinetic components indicate that the rate of bFGF association to the low and high affinity sites is comparable, but the rate of bFGF dissociation from the cell surface receptor is It has been shown to be 23 times slower than the associated heparan sulfate (Nugent & Edelman (1992) Biochemistry 31 : 8876). However, a slow dissociation rate was observed only when the receptor was bound to the cell surface, suggesting that simultaneous binding to both sites contributed to the overall high affinity binding. This may be possible considering the observation that the heparin binding site and the receptor binding site are located in separate regions near the molecule, as determined from the recently solved X-ray crystal structure of bFGF. (Zhang et al. (1991) Proc. Nat1. Acad. Sci. USA 88 : 3446; Eriksson et al. (1991) Proc. Natl. Acad. Sci. USA 88 : 3441; Ago et al. (1991) J. Biochem. 110 : 360; Zhu et al. (1991) Science 251 : 90).

The idea that bFGF antagonists have useful drug applications is not novel (reviewed in Gospodarowicz (1991) supra). It is now known that bFGF plays a central role in the development of smooth muscle cell lesions following vascular injury (Reidy et al. Circulation, Supplement III 86 : III-43). Overexpression of bFGF (and other members of the FGF family) correlates with many malignancies (Haraban et al. (1991) Ann. NY Acad. Sci. 638 : 232; Takahashi (1990) Proc. Natl. Acad. Sci. USA 87 : 5710; Fujimoto et al. (1991) Biochem. Biophys. Res. Commun. 180 : 386), and recently, neutralizing active anti-bFGF antibodies have been It has been found to inhibit solid tumor growth in vivo by inhibiting tumor-related angiogenesis (Poly (Hori) et al. (1991) Cancer Res. 51 : 6180). What should be noted in this regard is a recent therapeutic test of suramin, a polysulfated naphthalene derivative having known antiprotozoal activity, as an antitumor agent. Suramin is thought to inhibit the activity of bFGF by binding to the polyanion binding site and disrupting the interaction with the growth factor receptor (Middaugh et al. (1992) Biochemistry 31: 9016; Ericsson. (1992) supra). In addition to having many side effects and substantial toxicity, suramin interacts with several other heparin-binding growth factors, thereby linking beneficial clinical effects to specific drug-protein interactions. (La Rocca et al. (1990) Cancer Cells 2 : 106). The anti-angiogenic properties of certain heparin formulations have also been observed (Folkman et al. (1983) Science 221 : 719; Krum et al. (1985) Science 250 : 1375), and these effects are probably at least partially , Based on the ability to interfere with these bFGF signals. Certain heparin fractions that contribute to bFGF binding are now partially elucidated (Ishai-Michaeli et al. (1992) Biochemistry 31 : 2080; Turnbull et al. (1992) J. Biol. Chem. 267 : 10337), but typical heparin formulations are heterogeneous in terms of size, degree of sulfation and iduronic acid content. In addition, heparin affects many enzymes and growth factors. Therefore, except for monoclonal antibodies, no specific antagonist of bFGF is known.

SUMMARY OF THE INVENTION The present invention includes methods for identifying and producing nucleic acid ligands, and nucleic acid ligands thus identified and produced. The SELEX method described above allows the identification of a single nucleic acid ligand or family of nucleic acid ligands for a particular target. The method of the present invention allows analysis of nucleic acid ligands or families of nucleic acid ligands obtained by SELEX to identify and produce improved nucleic acid ligands.

  The present invention also includes a method for determining the three-dimensional structure of a nucleic acid ligand. Such methods include mathematical modeling and structural modification of SELEX derived ligands. The present invention further includes a method for determining which nucleic acid residues are required to maintain the three-dimensional structure of the ligand and which residues interact with the target to promote the formation of a ligand-target binding pair. included. In one embodiment of the invention, nucleic acid ligands are preferred due to their ability to inhibit one or more biological activities of the target. In such cases, a method is provided for determining whether a nucleic acid ligand effectively inhibits a biological activity of interest.

In addition, methods of identifying stronger binding RNA ligands and smaller, more stable ligands for use in pharmaceutical or diagnostic purposes are also included in the present invention.
The present invention includes improved nucleic acid ligands for HIV-RT and HIV-1 Rev proteins. The invention also specifically includes nucleic acid sequences that are substantially homologous to the nucleic acid ligands identified herein and that have the ability to bind to substantially the same HIV-RT or HIV-1 Rev protein. .

  Also included within the scope of the present invention is a method of performing a continuous SELEX experiment to identify humiliated nucleic acid ligands. In particular, extended nucleic acid ligands for HIV-RT are disclosed. Nucleic acid sequences that are substantially homologous to the extended HIV-RT nucleic acid ligand and that have the ability to bind to substantially the same HIV-RT are also included in the present invention.

The scope of the present invention includes nucleic acid ligands for HIV-1 tat protein. More specifically, an RNA sequence that can bind to the tat protein has been identified. The nucleic acid ligand solutions shown in FIGS. 26 and 27 are included in the present invention.

Further, a) preparing a candidate mixture of nucleic acids; b) fractionating the members of the candidate mixture based on affinity for the tat protein; and C) amplifying the selected molecule and relative to the tat protein. Included in the present invention is a method for identifying a nucleic acid ligand and ligand solution for HIV-1 tat protein comprising the step of obtaining a mixture of nucleic acids enriched for nucleic acid sequences having a particularly high binding affinity.

  In addition, nucleic acid ligands for thrombin are included in the present invention. More specifically, an RNA sequence that can bind to thrombin has been identified. The present invention includes the nucleic acid ligand solutions shown in FIGS.

  Further, a) preparing a candidate mixture of nucleic acids; b) fractionating members of the candidate mixture based on affinity for thrombin; and c) amplifying the selected molecule to be relatively high to thrombin Included in the present invention is a method of identifying a nucleic acid ligand and ligand solution for thrombin comprising the step of obtaining a mixture of nucleic acids enriched for nucleic acid sequences having binding affinity.

  More specifically, the present invention includes RNA ligands for thrombin identified by the method described above, including the ligand shown in FIG. 29 (SEQ ID NO: 137-155). The invention further includes RNA ligands for thrombin that are substantially homologous to any particular ligand and have the ability to bind to substantially the same thrombin. The invention further includes RNA ligands for thrombin that have substantially the same structure as the ligands described herein and have the ability to bind to substantially the same thrombin.

  The present invention further includes nucleic acid ligands for bFGF. Specifically, RNA sequences that can specifically bind to bFGF are provided. The present invention includes the nucleic acid ligand sequences shown in Table II-IV (SEQ ID NO: 27-89).

  The present invention also includes bFGF nucleic acid ligands, which are bFGF inhibitors. Specifically, RNA ligands that inhibit bFGF binding to receptors are identified and described.

  The present invention further includes a) preparing a candidate mixture of nucleic acids; b) fractionating the members of the candidate mixture based on affinity for bFGF; and c) amplifying the selected molecule to produce bFGF. A method of identifying a nucleic acid ligand and ligand sequence for bFGF comprising the step of obtaining a mixture of nucleic acids enriched for nucleic acid sequences having a relatively high binding affinity is included.

  More specifically, the present invention includes RNA ligands for bFGF identified by the methods described above, including the ligands shown in Tables II-IV. The invention further includes RNA ligands for bFGF that are substantially homologous to any particular ligand and have the ability to bind to substantially the same bFGF. The invention further includes RNA ligands for bFGF that have substantially the same structure as the ligands described herein and have the ability to inhibit substantially the same bFGF.

  The invention also includes modified nucleotide sequences based on the nucleic acid ligand sequences identified herein and mixtures thereof. Specifically, RNA ligands with modified ribose and / or phosphate and / or base positions are included in the present invention to increase the in vivo stability of the RNA ligand. Other modifications to RNA ligands are encompassed by the present invention, including specific changes in the base sequence and addition of nucleic acid or non-nucleic acid moieties to the original compound.

Detailed Description of the Preferred Embodiments This application is an extension and improvement of the method for identifying nucleic acid ligands called SELEX. The SELEX method is based on US patent application Ser. No. 07 / 714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligand”, and “Systematic Evolution of Ligand by Exponential Enrichment”, June 1990. U.S. Patent Application No. 07 / 536,428 filed on May 11, and PCT Patent Application Publication No. WO 91/19813 published Dec. 26, 1991 under the title "Nucleic Acid Ligand". Yes. The entire text of these applications is specifically incorporated herein by reference, including but not limited to the definition and explanation of the SELEX method.

  This application includes methods for identifying and producing improved nucleic acid ligands based on the basic SELEX method. This application includes different sections encompassing the following embodiments of the invention: SELEX method; II. Techniques for identifying improved nucleic acid ligands after performing SELEX; III. Continuous SELEX experiment-walking; IV. Elucidation of ligand structure by covariance analysis; Elucidation of improved nucleic acid ligands of HIV-RT (Example 1); VI. Performing a walking experiment with HIV-RT nucleic acid ligands to identify extended nucleic acid ligands; and VII. Improved nucleic acid ligand for HIV-1 Rev protein (Example 2); Nucleic acid ligand for HIV-1 tat protein (Example 3); Nucleic acid ligand for thrombin (Example 4); and Nucleic acid ligand for bFGF (Example) Elucidation of 5).

  Improved nucleic acid ligands for HIV-RT and HIV-1 Rev proteins are disclosed and claimed herein. The present invention includes specific nucleic acid ligands identified herein. The range of ligands encompassed by the present invention extends to all ligands of HIV-RT and Rev proteins identified by the methods described herein. More specifically, the present invention is substantially homologous to the nucleic acid ligands identified herein and binds to HIV-RT or Rev proteins under physiological conditions and is substantially the same. A nucleic acid sequence having the ability of: Substantially homologous means that the degree of homology is 70% or more, most preferably 80% or more. Substantially homologous also includes 20 base pair flips in the region of the nucleic acid ligand that includes the base pairing region. Substantially the same ability to bind to HIV-RT or Rev protein means that the affinity is within two orders of magnitude of the affinity magnitude of the nucleic acid ligands described herein, and preferably one order of magnitude. Is within the range of the size of. It is known to those skilled in the art to determine whether a sequence is substantially homologous to the sequences identified herein and has substantially the same ability to bind to HIV-RT or Rev proteins. It is not particularly difficult for them.

I. SELEX method.
In its most basic form, the SELEX method is defined by the following sequence of steps:
1) Prepare a candidate mixture of nucleic acids of different sequences. A candidate mixture generally comprises a region of fixed sequence (ie, each member of the candidate mixture contains the same sequence at the same position) and a randomized sequence. Fixed sequence regions a) to aid in the amplification process described below; b) to facilitate the mimicry of sequences known to bind to the target; or c) for specific structural configurations in the candidate mixture Selected to increase the concentration of nucleic acid. Randomized sequences are either completely randomized (eg, the probability of finding a base at any position is a quarter) or only partially randomized (eg, the probability of finding a base at any position) Can be selected at any level between 0 and 100 percent).

  2) The candidate mixture is contacted with the selected target under conditions that favor binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acid of the candidate mixture is believed to form a nucleic acid-target pair between the target and the nucleic acid having the strongest affinity for the target.

  3) The nucleic acid with the highest affinity for the target is fractionated from the nucleic acid with the lower affinity for the target. In general, a significant amount (about 5-50%) of nucleic acid remains in the candidate mixture since only a few sequences (possibly one molecule of nucleic acid) corresponding to high affinity nucleic acids are present in the candidate mixture. As described above, it is necessary to set a standard for fractionation.

  4) During fractionation, the nucleic acid selected as having a relatively high affinity for the target is then subjected to a novel candidate mixture that is enriched in nucleic acids having a relatively high affinity for the target. It is amplified when it is generated.

  5) By repeating the fractionation and amplification steps described above, the proportion of unique sequences in the newly formed candidate mixture will gradually decrease and the average degree of nucleic acid affinity to the target will generally increase. In extreme terms, the SELEX method produces a candidate mixture containing one or a few unique nucleic acids that are nucleic acids from the original candidate mixture with the highest affinity for the target molecule.

  The SELEX patent application describes this method in great detail. Here, a target that can be used in this method; a method of preparing an initial candidate mixture; a method of fractionating nucleic acids in the candidate mixture; and a method of amplifying the fractionated nucleic acid to produce a concentrated candidate mixture Is also included. The SELEX patent application also describes ligand solutions obtained for many target species, including both proteins that are nucleic acid binding proteins and those that are not.

SELEX provides a high affinity ligand for the target molecule. This represents an unprecedented achievement in the field of nucleic acid research. The present invention relates to a method for taking a ligand solution by SELEX in order to develop a novel nucleic acid ligand having the desired characteristics. The features of interest for a particular nucleic acid ligand vary. All nucleic acid ligands can form complexes with the target species. In some cases, it is preferred that the nucleic acid ligand acts to modify one or more biological activities of the target. In other cases, the nucleic acid ligand serves to identify the presence of the target, and the effect on the biological activity of the target is irrelevant.
II. Technology for identifying improved nucleic acid ligands after SELEX.

  To produce a nucleic acid that is preferred for use as a drug, the nucleic acid ligand 1) binds to the target in a manner that can achieve the desired effect on the target; 2) is as small as possible to obtain the desired effect; 3) as stable as possible; and 4) not all turtles that are preferably ligands specific to the selected target, but in most situations the nucleic acid ligand has the highest possible affinity for the target. It is preferable to have properties. Modification or derivatization of ligands that provide resistance to degradation during treatment and in situ clearance, ability to cross various tissue or cell membrane barriers, or other additional properties that do not significantly interfere with affinity for the target molecule May be given as an improvement. The present invention includes a method for obtaining improved nucleic acid ligands after performing SELEX.

Measurement of ligand effect on target molecule function . One use of nucleic acid ligands obtained by SELEX is to find ligands that change the function of the target molecule. Since ligand analysis requires much more work than encountered during SELEX enrichment, it is a good method to first measure inhibition or enhancement of target protein function. Such functional testing of the combined ligand pool can also be performed prior to cloning and sequencing. Measurements of the biological function of the selected target are generally available and known to those skilled in the art and can be readily performed to determine whether inhibition occurs in the presence of a nucleic acid ligand.

Ligand affinity measurement . SELEX enrichment provides many cloned ligands with possibly different affinities for the target molecule. Sequence comparison may result in a consensus secondary structure and primary sequence that allows the classification of ligands into motifs. Although a single ligand sequence (with some variation) is often found in the entire population of cloned sequences, the degree of expression of a single ligand sequence in the sequence cloned population of ligands depends on the affinity of the target molecule Is absolutely uncorrelated. Thus, being abundant is not the only criterion for determining a “winner” after SELEX, but to weight the importance of consensus reached by sequence comparison, various ligand sequences (each motif found by sequence analysis) A binding measurement is required. The combination of sequence comparison and affinity measurement should lead to the selection of candidates for further expanded ligand characterization.

Information boundary measurement . An important approach to determine the amount of sequence corresponding to specific affinity is to establish the boundaries of that information in the ligand sequence. This is conveniently accomplished by selecting end-labeled fragments from the pool of hydrolyzed ligands of interest so that the 5 'and 3' boundaries of information can be found. To determine the 3 'boundary, in vitro large scale transcription of the PCR ligand is performed, the RNA is gel purified using UV shielding on an intensifying screen, the purified RNA is phosphatase treated and fully phenolic Extract, label with 32P with kinase, and gel purify the labeled product (use gel film as guide). The resulting product is then subjected to preliminary partial digestion with ribonuclease (RNase) T1 (at 50 ° C. in 7M urea, 50 mM sodium citrate, pH 5.2 buffer at different enzyme concentrations and times) and alkaline hydrolysis. (Adjust to pH 9.0 by premixing 1M bicarbonate and carbonate solution with 50 mM NaCO 3; test at 95 ° C. for 20 to 60 minutes). Once the optimal conditions for alkaline hydrolysis have been established (and thus equally distributed from small to large fragments), it can be scaled up to obtain sufficient material for selection by the target (usually with a nitrocellulose filter). The binding measurement is then assembled by changing the target protein concentration from the lowest saturated protein concentration to a protein concentration that binds about 10% of the RNA as measured by ligand binding measurement. Rather than reducing the absolute amount of target (if the target supply allows), the target concentration should be changed by increasing the volume; because the amount of RNA bound to the filter is limited by the absolute amount of target, This gives a good signal to noise ratio. The RNA is eluted as in SELEX and then run on a denaturing gel along with the T1 partial digest so that the position of the hydrolysis band can be related to the ligand sequence.

The 5 'boundary is determined similarly. Large scale in vitro transcripts are purified as described above. There is one way to label the 3 'end of RNA. One method ³² (to buy or ³² P-Cp) kinased the Cp at P and is bound to RNA purified with RNA ligase. The labeled RNA is then purified as described above and the same experiment is performed. Another method is partial alkaline hydrolysis of unlabeled RNA, annealing as a measure of band position, and extending the labeled primer with reverse transcriptase. One of the advantages over the pCp labeling method is the simplicity of the method, a more complete ladder sequencing that can be correlated with boundaries (by dideoxy chain end sequencing), and a higher amount of product that can be measured. . The disadvantage is that the elongation of the eluted RNA sometimes contains artificial stops, so it is controlled by spotting the starting material on a nitrocellulose filter without washing, measuring it as input RNA. It is important to.

As a result, it is possible to find the boundary of sequence information required for high affinity binding to the target.
A teaching example is the measurement of information boundaries found in HIV-RT nucleic acid ligands. (See US patent application Ser. No. 07 / 714,131 filed Jun. 10, 1991 and PCT patent application WO 91/19813 published Dec. 26, 1991 entitled “Nucleic Acid Ligand”. thing). These experiments are described in detail below. The original pool of concentrated RNA resulted in a small number of specific ligands to HIV-RT (one ligand 1.1 accounted for 1/4 of the total population and the nitrocellulose affinity sequence reduced 1/2. And a few RNAs had no affinity for either). Two high affinity RT ligands are sequence. . . UUCCNNNNNNNNNCGGGAAA. . . . (SEQ ID NO: 6) was shared. Boundary experiments for both ligands established a clear 3 'boundary and a slightly unclear 5' boundary. From the boundary experiment and the second SELEX experiment, the most high affinity ligand is the essential information UCCGNNNNNNNCGGGAAAAN'N'N'N '(SEQ ID NO: 7) (where N' is formed by pairing UCCG to CGGG) It is speculated that 5'U is not important for affinity even with small deletions. In this application, due to the construction of the model compound, only one 5'U sequence has no difference in affinity compared to that of one 5'U '(shared by comparison of one ligand); It was confirmed that removal of both U's resulted in a 5-fold decrease in affinity and subsequent removal of C resulted in a more dramatic loss in affinity. The 3 ′ boundary, which was clearly visible in the boundary experiment, was not very noticeable. This new information can be used to derive that what is important at the 3 ′ end is to have at least three base pair nucleotides (in a sequence that loops between one strand of axis 1). Only one base pair nucleotide results in a 12-fold affinity loss. In the absence of 3 ′ base pair nucleotides (when cut at the end of loop 2), the affinity is reduced by approximately 70-fold.

Quantitative and qualitative assessment of individual nucleotide contributions to affinity.
Secondary SELEX . Once the minimal high affinity ligand sequence is identified, it is useful for identifying nucleotides within critical boundaries for interaction with the target molecule. One method is to create a new random template in which all nucleotides of the high affinity ligand sequence are partially randomized, or completely random blocks are interspersed in blocks with different randomizations. . Such “secondary” SELEX generates a pool of ligand sequences in which critical nucleotides or structures are absolutely conserved, less critical features are preferred, and unimportant positions are unbiased. . Secondary SELEX thus helps to further elaborate consensus based on a relatively small number of ligand sequences. Furthermore, even high affinity ligands with sequences that are undecided in the original SELEX may be provided.

  In this application we show such biased randomization of the ligand for HIV-1 Rev protein. US Patent Application No. 07 / 714,131 filed June 10, 1991 and PCT patent application WO 91/19813 published December 26, 1991 entitled "Nucleic Acid Ligand" 1 Nucleic acid ligands for Rev protein have been described. One of these ligand sequences bound with higher affinity than all other ligand sequences (Rev ligand sequence 6a shown in FIG. 12), but only one in 53 isolates cloned and sequenced. Existed as a copy of. In the present application, each nucleotide of the 6a sequence (limited to the sequence of the portion consisting of the Rev protein binding site defined by homology to the other of Rev ligand motif I) is transferred to the other during oligonucleotide synthesis. This sequence was incorporated into a secondary SELEX experiment mixed with 3 nucleotides in a ratio of 62.5: 12.5: 12.5: 12.5. For example, when this sequence is incorporated at position G1 during oligo synthesis, the G, A, T, and C reagents are mixed in a ratio of 62.5: 12.5: 12.5: 12.5. Ligand was cloned from this mixture so that a more comprehensive consensus description was obtained after 6 SELEX using Rev protein.

Nucleotide substitution . Another method is to test oligo-transcribed variants when the SELEX consensus is confused. As mentioned above, this helped to understand the nature of the 5 ′ and 3 ′ boundaries of the information needed to bind HIV-RT. As shown in the appended examples, this helped to quantify nucleotide consensus within axis 1 of the false knot in HIV-RT.

Chemical modification . Another set of useful techniques is described comprehensively as chemical modification experiments. Such experiments are used to explore the non-denaturing structure of RNA by comparing the modified pattern with the non-denatured one. The chemical modification pattern of the RNA ligand that is then bound to the target molecule is different from the non-denaturing pattern, indicating a structural change due to target molecule binding, or group protection by the target molecule. Furthermore, the target molecule does not bind to the RNA ligand when modified at a position critical to the binding structure of the ligand or at a position critical to interaction with the target molecule. Such experiments in which these positions are identified are described as “chemical modification interference” experiments.

There are a variety of available reagents known to those skilled in the art for performing such experiments (see Ehresmann et al. (1987) Nuc. Acids. Res. 15 : 9109). Chemicals that modify the base can be used to modify the ligand RNA. The pool of ligand binds to the target at various concentrations, the bound RNA is recovered (mostly as in border experiments), and the eluted RNA is analyzed for modification. Measurements are by sequential modification-dependent base elimination and aniline cleavage at base-free positions, or by reverse transcription assays at sensitive (modified) positions. In such a measurement method, a band (indicating a modified base) in unselected RNA that disappears relative to other bands in the RNA selected with the target protein appears. Similar chemical modification with ethyl nitrosourea, or mixed chemical synthesis, or enzymatic synthesis, for example with 2'-methoxy or phosphorothioate of ribose, used to identify essential groups on the basic skeleton it can. In experiments using 2'-methoxy versus 2'-OH mixtures, the presence of the essential OH group results in increased hydrolysis with respect to other positions of the molecule that are strictly selected by the target.

  An example of the use of chemical modifications to obtain useful information about the ligand and to help endeavor to improve its functional stability is given below for HIV-RT. The ethylnitrosourea modification interference identified five positions where the modification prevented binding and one of these positions that dramatically prevented it. Modifications of various groups on the base of the ligand have also been identified as critical to the interaction with HIV-RT. These positions were mainly in the 5 'helix and in the bridging loop sequence (axis I and loop 2 in Figure 1) that is highly conserved in SELEX phylogeny. These experiments not only confirmed the effectiveness of phylogeny, but also provided information on ongoing attempts to make more stable RNA. RT ligands with 2'-methoxy at all positions in the ribose part of the basic skeleton were synthesized. The bound molecule dramatically decreased its affinity for HIV-RT. Based on previous modification interference experiments and SELEX phylogenetic comparisons, it was determined that the 3 'helix (axis II in Figure 1) is essentially a structural component of this molecule. A ligand is then synthesized in which the 12 ribose i residues of the helix are 2'-methoxy, which binds any of the 6 remaining 14 residues 2'-OH bound to HIV-RT with high affinity. In order to investigate whether it is particularly necessary, all ribose spurious molecules were synthesized with mixed equimolar (empirically determined optimal) reagents for 2'-OH and 2'-methoxy formation. . After selection of HIV-RT from this mixture, alkaline hydrolysis reveals an enhanced hydrolysis band indicating that the 2 'hydroxyl is dominant at these positions. Analysis of this experiment led to the conclusion that residues (G4, A5, C13 and G14) must have 2'-OH for high affinity binding to HIV-RT.

  A comparison of the intensity of the bands of bound and unbound ligand may reveal not only modifications that interfere with binding, but also modifications that enhance binding. Exactly this modification creates a ligand and tests for enhanced affinity. Thus, chemical modification experiments are a way to explore additional local contact with the target molecule, just as “walking” (see below) is no additional nucleotide level contact with adjacent domains. It is possible.

  One of the products of the SELEX method is a consensus of primary and secondary structures that allows chemical or enzymatic synthesis of oligonucleotides designed based on that consensus. Such a ligand incompletely fills the available atomic space on the binding surface of the target molecule, since the SELEX replication mechanism requires a somewhat limited variation at the subunit level (eg, ribonucleotides). However, these ligands can be thought of as high affinity scaffolds that can be derivatized to make additional contact with the target molecule. Furthermore, the consensus contains group descriptors that are directly related to bonding and group descriptors that match the related group interactions. For example, each ribonucleotide of the false-knot ligand of HIV-RT contains a 2 'hydroxyl group on the ribose, but only one of the ribose of the false-knot ligand cannot be substituted with 2'-methoxy at this position. Similar experiments using a deoxyribonucleotide mixture with a ribonucleotide mixture (as done with a 2'-methoxy and 2 'hydroxy mixture) reveal ribose and its number that are not important for HIV-RT binding. Similar experiments with more extreme substitutions at the 2 'position also reveal the permissible substitutions at the 2' position. By this method, it is expected that derivatives of false knot ligands that give high affinity for HIV-RT can be found. Such derivatization does not preclude the use of cross-linking agents that specifically allow direct covalent binding to the target protein. Such derivatization analysis is not limited to the 2 'position of ribose, but includes derivatization of the nucleotide ligand at the base or at any position of the backbone.

  A logical extension of this analysis leads to the situation where one or a few nucleotides of a polymeric ligand are used as sites for chemical derivative search. The remainder of the ligand helps to properly tether the monomer (or group of monomers) to be tested for non-interfering binding and increased affinity. Such a search will yield a small molecule that mimics the structure of the original ligand framework and has a significant and specific affinity for a target molecule independent of the nucleic acid framework. These derivatized subunits, which have advantages over the original SELEX ligand in terms of mass production, clinical route of administration, in vivo delivery, clearance or degradation, can become therapeutic agents and traces of the original ligand Leave almost no. In other words, this approach is an additional utility of SELEX. SELEX ligands allow chemical exploration directed to defined sites on target molecules known to be important for target function.

Structure determination . These efforts helped to identify and evaluate the ligand sequence and structure-dependent association for HIV-RT. Another method is implemented to allow analysis at the atomic level of the ligand / target molecule complex. These are NMR spectroscopy and X-ray crystallography. With such a structure in hand, a rational design can then be made as an improvement on the evolved ligand supplied by SELEX. Computer modeling of the nucleic acid 20 structure is described below.

Chemical modification . The present invention includes a number of chemically modified nucleic acid ligands to enhance the in vivo stability of the ligand or to facilitate or mediate in vivo delivery of the ligand. Examples of such modifications include chemical substitutions at the ribose and / or phosphate positions of specific RNA sequences. See, for example, Cook et al., PCT application WO 9203568; Shinazi et al., US Pat. No. 5,118,672; Hobbs et al. (1973) Biochem. 12 : 5138; Guschlbauer et al. (1977) Nucleic Acids Res. 4 : 1933; Shibaharu et al. (1987) Nucl. Acids Res. 15 : 4403; Pieken et al. (1991) Science 253 : 314, each of which is specifically incorporated herein by reference.

III. Continuous SELEX Experiment-Walking Method In one embodiment of the present invention, after the minimum consensus ligand sequence is determined for a target, a random sequence is added to the minimum consensus ligand sequence to further (possibly separate) the target. Can be contacted with adjacent domains). This method is called the “walking method” in the SELEX patent application. Examples of successful use of the walking protocol to develop ligands with enhanced binding to HIV-RT are shown below.

  The walking method experiment includes one SELEX experiment that is performed sequentially. A new candidate mixture is generated such that each member of this candidate mixture has an immobilized nucleic acid region corresponding to a SELEX-derived nucleic acid ligand. Each member of the candidate mixture also contains a randomized region of sequence. This method makes it possible to identify what are termed “extended” nucleic acid ligands, which contain regions that bind to one or more binding domains of a target.

IV. Elucidation of Ligand Structure by Covariance Analysis The present invention includes a computer modeling method for determination of the structure of a nucleic acid ligand, as well as an empirical method for determination of the three-dimensional structure of a nucleic acid.

  Secondary structure prediction is a useful guide for correcting sequence alignment. This is also a useful tool for correcting the 3D structure prediction by forcing some bases into the outline of the A-type helix.

  There is a table of energy parameters for calculating the stability of the secondary structure. Early secondary structure prediction programs attempted to simply maximize the number of base pairs formed by the sequence, but most recent programs have the lowest free energy calculated by these thermodynamic parameters. Trying to find the structure. There is one problem with this approach. First, thermodynamic rules are inherently inaccurate (typically up to 10%) and there are many different possible structures within 10% of the overall minimum energy. Second, the actual secondary structure depends on sequence folding and synthesis dynamics and is not present in the overall minimum energy state. Nevertheless, for short arrays, these warnings are not as important, since the possible structures that can be formed are very limited.

  The brute force prediction method is a dot-plot: make an N × N plot of the sequence against itself and mark all X where possible with base pairing. X skews indicate possible spiral positions. A comprehensive branching search method then searches for all possible forms of compatible (ie, non-overlapping) spirals of length L or longer; Rank the degree of gender. The advantage of this method is that all possible shapes (topologies) including false knot placement are tested and some suboptimal structure is automatically generated. The disadvantage of this method is that, in the worst case, it works in proportion to the exponential factor of the size of the array and does not look deep enough to find the overall minimum (the size of the array and the actual used). Depending on the branching search method).

A clever (and currently the most used) prediction method is the Zuker program (Zuker (1989) Science 244 : 48). Based on an algorithm originally developed by Ruth Nussinov, the Zooker program makes a large simplification assumption that false knot placement is not allowed. This allows the use of a dynamic programming method that operates at times proportional to as little as N ³ to N ⁴ (where N is the length of the sequence). This Zooker program has become the most commonly used by biologists because it is the only program that can strictly handle sequences of hundreds of nucleotides or more. However, in several different SELEX experiments, the Zooker program is unable to predict false knot placement in that some false knotted RNA structures were obtained (and it was recognized by the eye). That is a fatal defect. You must use a brute force method that can predict false knot placement.

  A major element of the comparative sequence analysis of the present invention is sequence covariance. Covariance is when the body of one position is dependent on the body of another position; for example, the required Watson-Crick base pair is that one information of one position is the absolute value of the other position. Show strong covariance in terms of providing information. Covariance analysis was previously used to predict the secondary structure of RNA in which there are many related sequences with a common structure (eg, tRNA, rRNA, and group 1 introns). It became clear that covariance analysis can now also be used for third order contacts.

  Stormo and Gutell designed and implemented an algorithm that accurately measures the amount of covariance between a single position in an aligned set of sequences. This program is called “Mixie” (MIXY) (Mutual Information at position X and Y).

Consider an ordered set of sequences. At each column or position, the frequency of occurrence of A, C, G, U, and gap is calculated. This is called the frequency f (b _x ), that is, the frequency of the base b in the column x. Now consider one column at a time. The frequency that a certain base b appears in the column x is f (b _x ), and the frequency that the certain base b appears in the column y is f (b _y ). If If position x and position y is not related to the other (i.e., these positions are independent), the covariance is not the frequency of observing bases b _x and b _y the position x and y in any sequence, f (B _x b _y ) = f (b _x ) f (b _y ) should be _satisfied . These locations are said to be covariance if the observed pair frequency has a substantial deviation from the expected frequency. The amount of deviation from the expected value is quantified by the information measure M (x, y) (mutual information of x and y):

  M (x, y) is described as the number of bits of information regarding the position y because it recognizes the position y because it recognizes the main body of the position x. If there is no covariance, M (x, y) is zero; large values of M (x, y) indicate strong covariance.

  When this method was applied to a tRNA sequence dataset, these numbers correlated very well with the probability of intimate physical contact in the tertiary structure. The secondary structure is very obvious as a peak of M (x, y) value, and many tertiary contacts known from the crystal structure also appear as peaks.

These covariance values may be used to develop 3D structure predictions.
In some respects, this problem is similar to that of NMR structure determination. Finally, unlike crystallography, where an actual electron density map is obtained, NMR gives a series of interatomic distances. Depending on the number of interatomic distances obtained, one, a few, or many 3D structures will appear that match. In order to convert the matrix of interatomic distances into a 3D space structure, it was necessary to develop a mathematical method. One major technique used is distance geometry and controlled molecular dynamics.

  Distance geometry is a more formal and purely mathematical technique. The interatomic distance can be considered as coordinates in an N-dimensional space (where N is the number of atoms). In other words, the “position” of an atom is specified by N distances to all other atoms, instead of the three (x, y, z) we normally consider. The interatomic distance between all atoms is recorded in an N × N distance matrix. A complete and accurate distance matrix is easily converted to 3 × N Cartesian coordinates using matrix algebra operations. The basics of distance geometry applied to NMR is to handle incomplete data (only a few interatomic distances are known) and inaccurate data (the distance is known to an accuracy of at most a few angstroms). . Thus, much time for structure calculations based on distance geometry is spent on preprocessing the distance matrix, calculating the range of unknown distance values based on the known ones, and limiting the range of known ones. The Usually, a large number of structures were extracted from the distance matrix that matched the series of NMR data; if they all overlapped well, this data was sufficient to determine a unique structure. Unlike NMR structure determination, covariance only gives inaccurate distance values, but this is more promising than absolute knowledge as to whether certain distance constraints apply.

  Controlled molecular dynamics is a more specific method. In an experimental force field that attempts to describe the forces felt by all atoms (van der Waals forces, covalent bond length and bond angle, electrostatic force), all primordial random velocities (from Boltzmann distribution at a certain temperature) ) And calculate the motion of each atom in femtoseconds using the Newtonian kinetic equation, which simulates the molecular motion of many femtosecond time steps; this is “molecular dynamics”. In controlled dynamics, an extra special force is assigned to an atom when it exceeds a certain distance range.

  In this case, it is fairly easy to handle the nature of the probability of data using controlled molecular dynamics. Covariance values are translated into artificial force control over a range of distances between certain atoms; changing the force magnitude according to the covariance magnitude.

  NMR and covariance analysis generate distance constraints between atoms or positions, which are easily converted into structures by distance geometry or controlled molecular dynamics. Another source of experimental data utilized to determine the three-dimensional structure of nucleic acids is chemical or enzymatic protection experiments, which generate solvent access restrictions on individual atoms or positions.

V. Elucidation of Improved Nucleic Acid Ligand for HIV-RT An example of the method of the present invention is provided herein for the nucleic acid ligand of HIV-1 reverse transcriptase (HIV-RT). U.S. Patent Application No. 07 / 714,131 and PCT Patent Application Publication No. WO 91/19813 published December 26, 1991 entitled "Nucleic Acid Ligands" include SELEX on HIV-RT targets. The results obtained are described. By examining nucleic acid sequences that were found to have high affinity for HIV-RT, it was concluded that the nucleic acid ligand solution was arranged as a false knot.

  Described herein is an experiment that establishes the minimum number of sequences necessary to represent a nucleic acid ligand solution by boundary examination. Also described is the construction of ligand solution variants that are used to assess the contribution of individual nucleotides in solution to binding of the ligand solution to HIV-RT. 1) to confirm the predicted false knot structure; 2) to determine which modifiable groups are protected from chemical attack (or become unprotected by conjugation) when attached to HIV-RT. And 3) ligands to determine what modifications interfere with binding to HIV-RT (possibly due to a three-dimensional modification of the ligand solution) and are therefore likely involved in proximity contact with the target. A chemical modification of the solution is also described.

  The previously determined nucleic acid ligand solution is shown in FIG. Axis 1 (labeled) is conserved and axis 2 is a relatively non-conserved RNA false knot (where X is non-conserved and X 'base pairs with X). In the first SELEX consensus, U1 was preferred (11 out of 18 sequences contributing to the consensus are in this relative position), but A1 is often found (6 out of 18 sequences). It was. There was one sequence of G4-C13 base pairs with CG substituted and AU substituted. The preferred number of nucleotides linked to the two strands of axis 1 was 8 (8 out of 18 sequences). The number and pattern of nucleotides forming the base pair consisting of axis 2 and the selection of A5 and A12 were obtained from a secondary SELEX consensus in which random regions were constructed as follows: NUNUCCGNNNNNNNNCGGGAAAANNNN (SEQ ID NO: 8 (N is randomized). One of the ligands was found to significantly inhibit HIV-RT and not AMV or MMLV reverse transcriptase.

Improvement of Information Boundary The first two SELEX experiments in which 32 nucleotide positions were randomized gave a high-affinity ligand with variable length at the 5 'end of axis 1; UCCG that can form a base pair, UCCG that can form a base pair with CGGG, or CCG that can form a base pair with CGG. Determination of sequence boundaries that confer high affinity for interaction with HIV-RT could be achieved by selection from partially alkaline hydrolysates of end-labeled cloned RNA. This is because the high affinity ligand is essential information UCCGNNNNNNNNCGGGAAAANN'N'N '(SEQ ID NO: 7) (where N' is the N in the 8-base loop sequence of the hairpin formed by pairing UCCG to CGGG) This is a rapid and qualitative analysis that suggests that the loss of affinity will be small even without 5'U. In order to more closely test the 5 ′ sequence in a uniform situation, the binding experiment shown in FIG. 2 was performed. The RNA transcribed from the oligonucleotide template was identical to the complete sequence shown in the upper right corner of the figure, except that the 5 'end was changed as shown in the squares AE aligned at the left end. As a result, one 5′U is sufficient for the highest affinity binding to HIV-RT (squares A and B), binding is reduced without squares (square C), and an additional 5 ′ sequence. Removal of the sequence reduced the binding just like the non-specific sequence (square D). For the remainder of the experiments described herein, a design with only one 5′U (U1) (hereinafter referred to as Ligand B) was used.

  The dependence on the length of axis 2 was also tested by making various 3 'sections at the 3' end of ligand B. Deletion of 3 nucleotides (A24-U26) from the 3 'end did not change the affinity of the molecule for HIV-RT. Deletion of 4 nucleotides at the 3 ′ end (C23-U26) reduces binding by 7-fold, 5 (G22-U26) reduces by approximately 12-fold, and 6 nucleotides (U21-U26, ie 3 ′ helix) None) resulted in a 70-fold decrease in affinity. Such a drop is less dramatic than the drop found with the single base substitution reported below, which is (along with other data reported below) that this helix is primarily in loop 2. It suggests that it plays a structural role in supporting the positioning of very important groups.

SELEX consensus test on axis 1 . Various nucleotide substitutions in conserved axis 1 were prepared and their affinity for HIV = RT was measured. As shown in FIG. 3, there was little change in the affinity of the model RNA for U1A-substituted Korean HIV-RT. C at this position (which enhances axis 1 stability) or G (represented by the U-deletion experiment described above) resulted in an approximately 20-fold decrease in affinity. Replacement of G16 with A (which forms a base pair with U1) destroyed specific binding. Using a GC pair instead of C2-G15 also broke the binding, and using it instead of C3-G14 reduced the binding by a factor of about 10. One of these positions was highly conserved in the phylogeny of SELEX ligands. Various combinations replaced G4-C13 base pairs. These orders that affected the affinity were: G4-C13 = CG>UA> AU >>>> AC (where AU is about 20 times more affinity than G4-C13). And AC was reduced at least 100 times). These results are consistent with the previously measured SELEX consensus.

Chemical search for false knot structure . To explore the native structure of ligand b, to identify chemical modifications that significantly reduce the affinity of ligand B for HIV-RT, and to discover structural changes associated with binding by HIV-RT, many A chemical modification experiment was conducted. The chemicals used were ethylnitrosourea (ENU) that modifies phosphoric acid, dimethyl sulfate (DMS) that modifies the base pairing surface of C (at N3) and A (at N1), U (at N3) and a small amount. Carbodiimide (CMCT) that modifies the base-pairing surface of G (at N1), diethylpyrocarbonate (DEPC) that modifies A7 and a small amount of G7, and modifies the N1 and N2 base pairs of G It is ketoxal. Most chemical modification measurements were made with ligand B sequences that were elongated to include sequences that could be annealed with labeled primers and extended with AMV reverse transcriptase. Measurements of positions modified with ENU or DEPC were performed on ligand B using each modification-dependent hydrolysis or removal of the modified base followed by cleavage of the basic backbone at this site.

  The results of the search for the undenatured structure compared to the modification of the modified ligand B are summarized in FIG. The pattern of ENU modification is not different between the denaturated and undenatured states of the ligand, suggesting that the solution structure of the false knot has no stable involvement of the N7 position of phosphate or purine. . Other qualification data. Axis 2 is rather stable and resistant to any chemical modification affecting the indicated base pairing, but the terminal A6-U26 is between the base paired state and the denatured state at this position. It suggests some sensitivity to modifications that show equilibrium. Single-stranded A (A5, A17, A18, A19, and A20) is fully reactive with DMS, while A5, A19, and A20 have reduced reactivity to DEPC. Axis 1 base pair is G4-C13> C3-G14> C2-G15> U1-G16 (where G4-C13 is completely resistant to chemical modification and U1-G16 is highly reactive) It seems to express the order of resistance to modification. This suggests that this small helix of the false knot has undergone a transient directional degeneration, or “fraying”.

Protection of ligand B from chemical modification by HIV-RT . As shown in FIG. 5, protein binding alters the flaring properties of helix 1 depending on whether it is stabilized or protected. Originally reactive U1 is also protected by binding. Protein binding enhances the sensitivity of base pair A6-U26, suggesting that it is not paired in the bound state. This can be because the non-denatured knot knot recognized by RT cannot bridge the axis 1 to the end of the axis 2 or by changing the arrangement from the non-denatured state, coupling increases the length requirement for loop I. This indicates that the length of the single-stranded nucleotide loop I during binding is insufficient. Loop II A17 and A19 are also protected by binding to HIV-RT. In addition, A12 of the single-stranded base bridge is protected by a bond.

Examination of RT ligand B modification interference . RNA ligand B was partially modified (with all chemicals described above for structure determination). This modified population bound to various concentrations of protein and measured the modified position of the binding species. From here it can be determined where the modification interferes with binding and where the modification has no or little effect. A schematic summarizing these modification interference results is shown in FIG. As shown here, much of the significant hindrance to binding is concentrated on the left side of the fake knot containing axis 1 and loop 2. This is also part of a molecule that was highly conserved (primary sequence) in the set of sequences isolated by SELEX, and this is the most dramatic binding affinity for HIV-RT by substitution experiments. It has just dropped.

Replacement of the 2′-hydroxyl on the ribose of ligand B with 2′-methoxy . The “rRNA” molecule, in which 2′-methoxy is attached to the 2 ′ carbon of ribose instead of the normal hydroxyl group, is resistant to enzymatic and chemical degradation. To test how extensively 2′-methoxy can replace 2′-OH in RT ligands, four oligos were prepared as shown in FIG. Since the ligand completely substituted with 2'-methoxy hardly binds (ligand D), and it was found that most of the modification interference sites are concentrated at one end of the false knot. Were limited to the non-specific 3 'helices shown in squares B and C. Both of these ligands bind to HIV-RT with high affinity. Next, as shown in FIG. 7C, all 2′-methoxy substitutions allowed for the ribose on axis 2 are mixed with 2′-methoxy and 2′-OH bosphoramidite reagents at the remaining 14 positions. Were prepared. These oligos were selected by HIV-RT, followed by alkaline hydrolysis and gel separation of the selected RNA (2′-methoxy form is not involved in alkaline hydrolysis unlike 2′-hydroxyl form). . As determined by visual inspection of the film (see FIG. 8) and quantitative measurement of relative intensity using an ambis detection system (see comparative method in the following examples), they were mixed and incorporated. Ligands selected by HIV-RT from the population show a marked increase in hydrolysis at the C13 and G14 positions, indicating interference by 2'-methoxy at these positions. In related experiments where all positions were analyzed in this manner, G4, A5, C13 and G14 showed 2'-O-methyl interference.

  The results of substitution experiments, quantitative boundary experiments, and chemical exploration experiments gave much information about the nature of HIV-RT's false knot inhibitor and highlighted the critical contact region on this RNA. These results are given for each of the following nucleotides:

  Substituting U1 for A causes little loss of affinity, but is different for C or G. U1 probably generates a temporary base pair with G16, but modification of U1-N3 with CMCT does not interfere with binding to HIV-RT. However, binding by HIV-RT protects N3 from U1, possibly by steric or electrostatic shielding at this position. Substitution with C, which forms a more stable base pair with G16, reduces affinity. Replacement of G16 with A to form a stable U1-A16 pair destroys specific affinity for HIV-RT, and modification of G16-N1 strongly interferes with binding to HIV-RT. This modification of G16-N1 must prevent very important contact with the protein. It is not clear why G substitution for U1 reduces affinity and does not allow A substitution. The G substitution is apparently an affinity that is equivalent to an in vitro transcript with an extra G at the 5 'end, although the 5' end of the RNA is one nucleotide shorter but U1 is the 5 'end nucleotide. (Fig. 7). Probably A at U1 replaces a possible U interaction with a similar or different interaction with HIV-RT, which is an impossible substitution with C or G at this position.

  The next base pair on axis 1 (C2-G15) cannot be replaced by a GC base pair without a complete loss of specific affinity for HIV-RT. Modification of the base-pairing surface of either nucleotide strongly prevents binding to HIV-RT, and binding to HIV-RT protects against these modifications. When the next base pair C3-G14 is replaced with a GC pair, the loss of affinity is not so dramatic, but modification of this position strongly interferes. Replacement of G4-C13 with a CG pair does not affect binding, and replacement with a less stable AU and UA pair allows for somewhat specific affinity. Replacing these positions with non-base-paired A-C destroys specific binding. This is the appearance of a CG substitution and one AU substitution in the first SELEX phylogeny at this position, the non-reactivity of this base pair in the native state, and the high degree of modification found in these bases. Correlate with interference.

  The chemical modification data for Loop 2 fully guarantees the phylogenetic preservation seen in the first SELEX experiment. A strong modification hindrance is seen at positions A17 and A19. Weak modification interference occurs at A20 which correlates with some loop 2 findings of the first SELEX deleted at this relative position (however, the chemical interference experiments performed were all possible that the base could make with HIV-RT. Have not been tested for complete contact). A18 is not conserved in the original SELEX and does not interfere with modification at this position, and this position is not protected from modification by binding to HIV-RT.

  Combined with the above data, the essential components of axis 1 are a single stranded 5 ′ nucleotide (U or A) that specifically contacts the protein with a protein and three base pair helices (C2-G15, C3-G14, G4-C13) (where the first one base pair has sequence-specific interaction with HIV-RT and the G4-C13 third loop closes at a strong base pair (ie, CG or (G-C) has a priority). Loop 2 should be described more broadly as GAXAA (16-20) due to the single-stranded nature of G16, which interacts with HIV-RT, probably in a sequence specific manner like A17 and A19. . Axis 2 varies considerably in the pattern and number of base-pairing nucleotides, but requires a minimum of 3 base pairs in axis 2 to obtain maximum affinity from the 3 'deletion experiments reported herein. Can be hypothesized. At least one nucleotide is required for loop 1 of the bound ligand, within the range of 8 nucleotides that connect the single strands that make up the axis 1 helix.

  The revised HIV-RT ligand obtained based on the method of the present invention is described in FIG. The major differences between the one shown in FIG. 1 (which is based on the first and second order SELEX consensus) are the length of axis 2, the more retracted specification of base pair G4-C13, the size of loop 1 (this is the axis Directly related to the size of 2), and the single-stranded properties of U1 and G16.

How can these differences be harmonized? Without being limited by theory, the SELEX strategy requires 5 ′ and 3 ′ fixed sequences for replication. In any RNA sequence, such additional sequences enhance capacity with respect to other configurations that compete with the configuration of the high affinity ligand. As a result, additional structural elements that do not contribute directly to affinity (eg, stretched axis 2) are selected. If the first two base pairs of axis 1 must be CG for sequence specific contact, the most stable closed base pair is reselected to avoid configuration ambiguity. C13 (Freier et al. (1986) Proc. Natl. Acad. Sci. USA 83 : 9373). The sequence-specific selection of U1 and G16 is that they coincide with the ability to base pair; other nucleic acids such as Klenow fragment / primer-template conjugation and tRNA / tRNA synthetase. In the ligand-protein complex, there is a significant local denaturation of base pair nucleotides that may also occur in this case (Freemont et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 8924; Ronald ( Ronald) et al. (1989) Science 246 : 1135).

VI. Performing walking experiments with HIV-RT nucleic acid ligands to identify extended nucleic acid ligands .

The immobilization sequence (28 nucleotides) located 5 'of the false knot consensus ligand reduces the affinity for HIV-RT, and the immobilization sequence (31 nucleotides) added 3' to the ligand increases its affinity. Have already been found. Therefore, in order to determine if a higher affinity ligand consensus for HIV-RT could be obtained, a SELEX experiment was performed and a 30 nucleotide variable region was added 3 'to the ligand B sequence. Individual isolates were cloned and sequenced after the 16th round. This sequence is classified into one motif in FIG. 9 and listed (sequence ID number: 115-135). A schematic diagram of the secondary structure of each motif and the conservation of the primary sequence is shown in FIG. The distance between the RNase H of HIV-RT and the catalytic domain of the polymerase was recently 18 base pairs in a type A RNA-DNA hybrid (computed by computer) incorporated into a pocket of 3.5A resolution structure derived from X-ray crystallography. (Kohlstaedt et al. (1992) Science 256 : 1783). The distance of the base conserved in the false knot and extended ligand sequence from the base concentration site determined to be very important for this interaction is also about 18 base pairs. Thus, the false knot interacts with the catalytic site of the polymerase (wherein the ligand was shown to bind to HIV-RT lacking the RNAse H domain), and the evolved extension to the false knot is RNAse H It is concluded that it interacts with the domain. In general, the ligands tested from each motif enhance the affinity of ligand B for HIV-RT by at least 10-fold.

VII. Elucidation of improved nucleic acid ligands of HIV-1 Rev protein .
Examples of the method of the invention are provided herein for the nucleic acid ligands of HIV-1 Rev protein. US Patent Application No. 07 / 714,131 and PCT Patent Application Publication No. WO 91/19813 describe the results obtained when SELEX was performed on a Rev target. Examination of nucleic acid sequences found to have high affinity for Rev revealed that these sequences can be classified into three motifs (I, II, and III). The ligand for motif I is a composite of the individual motifs described by motifs II and III and generally appears to bind to Rev with higher affinity. One of the motif I ligand sequences (Rev ligand sequence 6a) bound with significantly higher affinity than all the cloned and sequenced ligands. As shown in FIG. 12, it is hypothesized that the 6a sequence forms a ridge between one helix and has a small number of base pairs across this ridge.

  The present specification identifies a proposed secondary structure, finds a place where Rev protein binding protects the ligand from chemical attack, and is designed to detect nucleotides essential for Rev interaction. The chemical modification experiment performed in 6a is described. In addition, a second SELEX experiment was performed with biased randomization of the 6a ligand sequence to more comprehensively describe the consensus for the highest affinity binding to the HIV-1 Rev protein.

Chemical modification of Rev ligand . Determine its possible secondary structural elements, find out which modifications interfere with ligand binding by Rev, identify which positions are protected from modification to protein binding, and In order to detect possible changes, a chemical modification study of Rev ligand 6a was performed.

  The chemicals for modification are ethyl nitrosourea (ENU) that modifies phosphoric acid, N3 at base pairing position C and N1 of adenine, dimethyl sulfate (DMS), and base pairing positions N1 and N2 of guanine. It includes ketoxal, carbodiimide (CMCT) that modifies the N3 position of the base pairing position of uracil and a small amount of guanine, and diethylpyrocarbonate (DEPC) that modifies the N7 position of adenine and a small amount of N7 of guanine. ENU modification is measured by modification-dependent hydrolysis of the labeled RNA strand, while all other modifiers are used on the extended RNA ligand and revealed by primer extension of the oligonucleotide annealed at the modified position Became.

The chemical search for the unmodified structure of the Rev ligand is summarized in FIG. Computer-predicted secondary structure (Zoker (1989), supra; Jaeger et al. (1989) Proc. Natl. Acad. Sci. USA 86 : 7706)) and the unmodified modification data are roughly consistent; It consists of two helical regions, one 4-base hairpin loop, and three “raised” regions (see FIG. 13 for the definition of these structural “elements”).

ENU modification of phosphate did not change the ligand under non-denaturing and denaturing conditions, indicating that the phosphate group was not involved in the secondary or tertiary structure of RNA. . In general, all computer predicted base pairing regions are protected from modification. One exception is a slight modification of N7 (G ¹⁰ , A ¹¹ , G ¹² ) in the central helix (usually the protected position in the helix). These modifications are probably the result of helical respiration; the absence of modification of the base-pairing surface of the central helix is due to the small helical distortion that the accessibility of N7 is rather than a complete local cleavage of the RNA I suggest that. The G19-U22 hairpin loop is fully modified except for some partial modifications of G19.

The most interesting regions of the undenatured structure are the three “raised” regions U8-U9, A13-A14-A15, and G26-G27. U8-U9 is fully modified by CMCT, which probably indicates the orientation of the base to the solvent. A13, A14, and A15 are all modified by DMS and DEPC, with the most potent modification occurring in the central A14. The bump opposite the A13-A15 region indicates complete protection of G26 by DMS, indicating a slight modification of A27. In another study of Rev binding RNA (Bartel 1 et al. (1991) Cell 67 : 529), the non-canonical bases of A: A and A: G corresponding to A13: A27 and A15: G26 of this ligand. Insist on the existence of pairing. Although this modification data does not rule out these possibilities, the equal stereo A: A base pair suggested by Bartel et al. Will use the NlA position for base pairing and will therefore be resistant to DMS treatment. Also, the A: G pair similarly uses either NlA or N7A for pair generation to make A resistant to DMS or DEPC.

Interfering with Rev binding modification . The results of the modification interference studies are summarized in FIG. 14 (quantitative data for individual modifiers is shown in FIGS. 15-19). In general, the modified binding interference between phosphate and base is concentrated in one region of the RNA ligand. As a first approximation, these regions correspond to one other motif present in the SELEX experiment prior to this study. Phosphate modification interference is probably the most probable actual site of ligand-protein contact and constitutes an additional criterion for classification into modification interference data areas.

The first region is concentrated in U24-G25-G26 and includes interference by phosphate, base-pairing surfaces, and N7 modification. These same three nucleotides conserved in the wild-type RRE were also tested in a modified interference study using a short RNA containing the RRE IIB axial loop (Kjems et al. (1992) EMBO J. 11 : 1119). It has been found to be very important for binding. Second region: The region is concentrated around G10-A11-G12, which also includes interference from phosphate, base-pairing surfaces, and N7 modifications. In addition, there is a smaller “mini region” surrounding C6-A7-U8, where phosphate and base-pairing surface modifications interfere with binding.

  Many base-pairing surface modifications throughout the ligand indicate interference with binding, presumably due to fluctuations in the secondary structure of the ligand. One “raised” base, U9 and A14, did not show modification interference, indicating that they have no role in specific base-pairing interactions / stacking or in contact with proteins. .

Chemical modification protection when RNA is bound to Rev. The “footprint” chemical modification data is summarized in FIG. Four positions, U8, A13, A15, and A27, when bound to Rev protein showed at least a 2-fold decrease in base pairing surface modification (and a similar decrease in N7 modification at the A position). A slight N7 modification of G10-A11-G12 under non-denaturing conditions was not detected when the ligand was modified in the presence of Rev. Unmodified G32 by chemical exploration of RNA undenatured structures, when complexed with Rev, exhibits strong modification of its base-pairing surface and N7 position. U31 and U33, 5 'and 3' of G32, show slight CMCT modification when the ligand binds to the protein.

Secondary SELEX using biased randomization of the mold . A template as shown in FIG. 21 was synthesized in which the Rev ligand 6a sequence was mixed with the other three nucleotides at each position in a ratio of 62.5 to 12.5 (6a sequence) to the other three nucleotides. . This biased template gave RNA with background affinity for the Rev protein (Kd = 10 ⁻⁷ ). The list of sequences shown in FIG. 21 was obtained by performing SELEX six times. The frequency distribution of nucleotides and base pairs found at each position, which is different from the distribution expected from the input distribution during template synthesis, is shown in FIGS. A new consensus based on these data is shown in FIG. The most striking differences from Rev ligand 6a are the relatively weak base pair A7-U31 substitution by GC pair, and U9 C by A14 U by U22 G by allowed or preferred substitution. is there. Absolutely conserved positions are at sites G10, A11, G12; A15, C16, A17; U24, G25; and C28, U29, C30. Although no substituted bases were found in G26 and A25, one and three deletions were found at these positions, respectively. One labeled transcript was synthesized, one with a sequence similar to the simple ligand 6a and the other replaced by the significant selection shown in FIG. These RNAs bound to the Rev protein in exactly the same way.

Many permutations of the axial region enhance its stability . Although there does not appear to be a significant selection of axes longer than 5 base pairs, this may be a necessary choice for replication (eg, ease of replication during the SELEX reverse transcription step). U.S. Patent Application No. 07 / 714,131 filed on June 10, 1991 and PCT Patent Application Publication No. WO 91/19813 published on December 26, 1991, in U9 Although there are some sparse substitutions of other nucleotides, this experiment shows a preferred substitution by C. The first SELEX also showed an A27 deletion. A surprising result is the frequent occurrence of C18-A pairing instead of C18-G23.

  The reason for the preference for this experiment which does not improve the measured binding affinity is because of the difference between SELEX binding reactions and these binding assays. In SELEX, a relatively enriched pool of heterogeneous RNA sequences (adjacent to essential fixed sequences) is bound to the protein. In the binding assay, a low concentration of uniform RNA sequence is bound. SELEX may choose for more discriminating placement certainty by increasing the probability of intermolecular and intramolecular contact with other RNA sequences. The priorities found in these secondary SELEX are suitable for clinical in vivo delivery of concentrated doses of RNA ligands and their modified homologues.

Nucleic acid ligand for HIV-1 tat protein . The present invention applies the SELEX method to the specific target HIV-1 tat protein. In the example dish 15 below, the experimental parameters used to isolate and identify a nucleic acid ligand solution for HIV-1 tat protein are described. FIG. 26 lists the nucleic acids sequenced after 10 iterations of the SELEX process.

FIG. 25 shows the naturally occurring TAR sequence that was found to be a natural ligand for the tat protein. The specific site of interaction between the tat protein and the TAR sequence was determined and is also described in FIG.

The sequence given in FIG. 26 is classified into three “motifs”. Each of these motifs represents a nucleic acid ligand solution for HIV-1 tat protein. The region of primary sequence conservation within each motif is surrounded by a dotted square. Motifs I and II contain a common structure that places conserved sequences (these sequences found in all or nearly all nucleic acid sequences that make up a motif) in the ridge adjacent to the elements of the helix. Primary sequence conservation (which is primarily in the single-stranded domain of each ridge) is also similar between motifs I and II. The third motif (III) is characterized by a large loop. These three motifs are represented schematically in FIG. There is no clear similarity between the nucleic acid ligands identified herein and the TAR sequences given in FIG.

Boundary analysis measurements were performed on one of the motif sequences of motif III. The boundary of recognition is shown in FIG. 26 with a solid line surrounded by a square. The boundary measurement was carried out by the method already described. Tuerk et al. (1990) J. MoI. Mol. Biol. 213 : 749; See Turk & Gold (1990) Science 249 : 505.

In FIG. 28, the binding affinities of sequences 7 (motif I), 24 (motif II), 29 (motif II), 31 (motif III) and the initial candidate mixture are shown. As can be seen, the members from each nucleic acid ligand solution motif have increased affinity for the tat protein compared to the candidate mixture of nucleic acids. Each ligand exhibits a significantly greater affinity for the tat protein than the TAR sequence.

  In order to produce the nucleic acid required for use as a drug, this nucleic acid ligand can: 1) bind to the target in a way that can exert the desired effect on the target; 2) as much as possible to obtain the desired effect. It is preferably small; 3) as stable as possible; and 4) a ligand specific for the selected target. In most, but not all cases, it is preferred that the nucleic acid ligand has the highest possible affinity for the target.

The present invention includes the specific nucleic acid ligand shown in FIG. 26 and the nucleic acid ligand solution shown schematically in FIG. The range of ligands covered by the present invention extends to all ligands for the tat protein identified by the SELEX method. More specifically, the present invention relates to 1) substantially homologous to the specific nucleic acid ligand shown in FIG. 26 and has the same qualitative binding ability to the tat protein, or 2) a substantially homologous to the nucleic acid ligand solutions shown in, substantially the same binding capacity for tat protein comprises a nucleic acid sequence. Substantially homologous means that the degree of homology of the primary sequence is 70% or more, most preferably 80% or more. Substantially the same binding ability for a tat protein means that its affinity is within two orders of magnitude of the affinity magnitude of the substantially homologous sequences described herein. It is not particularly difficult for those skilled in the art to determine whether a sequence (substantially homologous to the sequences described herein) has substantially the same ability to bind to the tat protein. Absent.

A summary of motifs I, II and III, and binding curves are shown in FIG. 28, indicating that sequences with little or no homology to the primary sequence have substantially the same ability to bind to the tat protein. If it is assumed that the ligands for each of these motifs bind to the same binding site of the tat protein, it is clear that binding is controlled by the secondary or tertiary structure of the nucleic acid ligand. Certain primary structures, represented herein by motifs I, II, and III, can assume a structure that is very similar to the binding site of the tat protein. For these reasons, this application binds to the tat protein, which has substantially the same structural type as the ligands provided herein and is substantially identical to the nucleic acid ligands shown in FIG. 26 or FIG. Nucleic acid ligands that have the capability are also included. A substantially identical structure includes all nucleic acid ligands having the common structural elements of motifs I, II and III that lead to affinity for the tat protein.

  The present invention also includes a ligand as described above, with certain chemical modifications added to increase the in vivo stability of the ligand or to facilitate or mediate in vivo delivery of ligand.

The nucleic acid ligands and nucleic acid ligand solutions for the HIV-1 tat protein described herein are useful as drugs and as part of gene therapy. By methods known to those skilled in the art, the nucleic acid ligand is introduced into cells infected with the HIV virus, where the nucleic acid ligand competes with the TAR sequence for the tat protein. Thus, transcription of the HIV gene is prevented.

Nucleic acid ligand for thrombin . The present invention includes the specific nucleic acid ligand shown in FIG. 29 (SEQ ID NO: 137-155). The range of ligands covered by the present invention extends to all ligands for thrombin identified by the SELEX method. More specifically, the present invention is a nucleic acid that is substantially homologous to the specific nucleic acid ligand (SEQ ID NO: 137-155) shown in FIG. 29 and has substantially the same binding ability to thrombin. Contains a ligand.

  The proposed structural summary shown in FIG. 30 for Group I and Group II ligands shows that sequences with little or no homology to the primary sequence still have substantially the same ability to bind thrombin. For these reasons, the present invention has substantially the same structure as the ligands provided herein, and has thrombin substantially the same as the RNA ligand (SEQ ID NO: 156-159) shown in FIG. Also included are RNA ligands that have the ability to bind. The “substantially identical structure” includes all RNA ligands having a common structural element of the sequence shown in FIG. 30 (SEQ ID NO: 156-159).

The present invention also includes the above-described ligands with certain chemical modifications to increase the in vivo stability of the ligand or to facilitate or mediate in vivo delivery of the ligand. In particular, the scope of the present invention includes thrombin RNA ligands containing 2′-NH ₂ modifications of ribose with RNA ligands.

The nucleic acid ligands and nucleic acid ligand solutions for thrombin described herein are useful as drugs and as part of gene therapy.
The concepts of vascular injury and thrombosis are important to understand the etiology of various vascular diseases, including the onset and progression of atherosclerosis, acute coronary syndrome, venous transplant disease, and restenosis after coronary angioplasty It is.

  The high affinity thrombin binding RNA ligand of the present invention is expected to have various properties. These features can be considered in line with recent understanding of hirudin peptide inhibitors and the structure and binding of thrombin. In line with this and without being limited to theory, RNA ligands are more likely to bind to a highly basic anionic outer site. In addition, there is a high possibility that RNA is not bound to a catalytic site having high specificity for a cationic arginine residue. The RNA ligand is expected to act in the same manner as the C-terminal hirudin peptide. Thus they do not strongly inhibit small peptidic substrates, but will inhibit fibrinogen clotting, protein C activation, platelet activation, and endothelial cell activation. If fibrinogen coagulation and TM binding activity can be separated at the anionic outer site, different high affinity RNA ligands can inhibit these activities separately. In addition, one activity may be selected to produce an anticoagulant that is more potent than procoagulant.

  The SELEX method for identifying ligands for a target was performed using human thrombin as a target and a candidate mixture containing 76 nucleotide RNA with a 30 nucleotide region: region of randomized sequence (Example IV). ). After 12 SELEX sequences of many selected ligands were determined, revealing the existence of one group of sequences with common elements of the primary sequence.

A dramatic change in the binding of the RNA population was observed after 12 doses of SELEX compared to large amounts of 30N RNA. Sequencing of large amounts of RNA after 12 times also showed non-random sequence aspects. RNA was reverse transcribed, amplified and cloned to sequence 28 individual molecules (FIG. 29). Based on primary sequence homology, 22 RNAs were classified as class I and 6 RNAs were classified as class II. Of the 22 sequences of class I, 16 (of which 8 are identical) contained the same sequence motif GGAUCGAAG (N) ₂ AGUAGGC (SEQ ID NO: 9), while the remaining 6 sequences were limited Contained regions contained 1 or 2 nucleotide changes, or slight variations from N = 2 to N = 5. This conserved motif changed its position in the 30N region. In class II, three of the 6 RNAs were identical and all contained the conserved motif GCGGCUUUGGGCGCCGUUGCUU (SEQ ID NO: 10), starting at the 3rd nucleotide from the end of the 5 ′ fixed region.

Three sequence variant RNA ligands from class I (6, 16, and 18) and one from class II (27) (identified by sequence order) were used for individual binding analysis did. Class I RNA takes clone 16 kD to about 30 ⁿ M to Examples, kD class II RNA clone 27 was approximately 60 ⁿ M.

  To identify the minimal sequence required for specific high affinity binding of a 76 nucleotide RNA containing a variable 30N region flanking the 5 'and 3' fixed sequences, 5 'and 3' boundary experiments were performed. For 5 'boundary experiments, the 3' end of the RNA was labeled and hydrolyzed to obtain a pool of RNA with various 5 'ends. For 3 'boundary experiments, the 5' end of RNA was labeled and hydrolyzed to obtain a pool of RNA with various 3 'ends. The required minimal RNA sequence was determined by RNA protein binding to nitrocellulose and identification of labeled RNA by gel electrophoresis.

  The 3 'boundary experiment resulted in the boundaries of each of the four sequences (SEQ ID NO: 156-159) shown in FIG. These boundaries were consistent across all protein concentrations. The 5 'boundary experiment yielded the boundary plus or minus one nucleotide shown in FIG. 31 except for RNA 16, which gave a large boundary at a low protein concentration. A possible secondary structure of a thrombin ligand based on these boundary experiments is shown in FIG. 30B.

For binding analysis, RNAqo corresponding to the smallest and largest hairpin of class I clone 16 (24 and 39 nucleotides) and the hairpin of class II clone 27 (33 nucleotides), synthesized or transcribed (see FIG. 30B) . Results, RNA27 hairpins (compare RNA33R in Figure 31C to RNA27 in Fig. 31A) of fixed and variable regions show that bind with all 72 nucleotides transcript equal affinity (kD of about 60 ⁿ M) having a. Meanwhile kD clone 16RNA hairpin class I was elevated in size from 30 ⁿ M to 200 ⁿ M.

Modification of 2NH2 monoribose of pyrimidine residues of RNA molecules has been shown to increase RNA stability (resistance to degradation by RNase) in serum by at least 1000-fold. Binding experiments using class I and class II 2NH ₂ -CTP / UTP modified RNAs showed a significant decrease in binding compared to unmodified RNA (FIG. 32). However, binding by large amounts of 3, ONRNA showed a slight increase in affinity when modified.

A ssDNA molecule with the 15 nucleotide consensus 5'-GGTTGGGTTGGTTGG-3 '(G15D) (SEQ ID NO: 1) has been shown to bind to human thrombin and inhibit fibrin clot formation in vitro (Bock et al. ( 1992), as described above). The results of a competition experiment for thrombin binding between G15D and the RNA hairpin ligand of the present invention are shown in FIG. At the beginning of these experiments A), ³² P-labeled G15D was used as a tracer with increasing concentrations of unlabeled RNA or unlabeled G15D. As expected, when G15D was used to compete for its own binding, the binding of labeled DNA was reduced to 50% at equimolar concentrations (1 μM) of labeled and unlabeled competitor DNA. Class I clone 16 synthetic RNAs 24 and 39 and class II clone 27 synthetic RNA 33 were able to compete for G15D binding at this concentration. In B), the high-affinity class II hairpin ^{RNA33 (kD ≒ 60 n M)} 32 and P-labeled, together with unlabeled RNA or unlabeled G15DDNA increasing concentrations (kD ≒ ²⁰⁰ n M), used as a tracer did. In these experiments, G15D can compete effectively with RNA33 at higher concentrations than the competition of RNA33 itself (change to the right of binding), which is expected when competing with 3-4 fold higher affinity ligands. Is Rukoto. Hairpin Class II RNA33 (kD of about ⁶⁰ n M) is the hairpin Class I RNA24 (kD of about ²⁰⁰ n M), was only weakly competitive, which might have some overlap, These single classes of RNA suggest high affinity binding to different but adjacent or overlapping sites. Since both these RNAs can compete for G15D binding, this DNA 15mer is probably bound to the region of overlap between class I and class II hairpins.

Cleavage of chromogenic substrate S2238 . Peptide chromogenic substrate S2238 (HD-Phe-Pip-Arg-pnitroaniline) (HD-Phe-Pip-Arg-pNA) (mold) in the presence and absence of RNA ligands of the present invention • The ability of thrombin to cleave Pharmacia) was measured. This cleavage reaction, thrombin ^{10 -8} M and RNA10 ^-8 M, thrombin ^{10 -9} M and RNA10 ^-8 M or thrombin ^{10 -8} M and RNA10 ^-7 M, RNA had no inhibitory effect (FIG. 34A ). These results suggest that the RNA ligand does not bind to the catalytic site of the enzyme.

Cleavage of fibrinogen into fibrin and coagulation formation . The ability of thrombin to catalyze clot formation by cleavage of fibrinogen into fibrin was measured in the presence and absence of the RNA ligands of the present invention. When RNA is that present in a concentration equal to Kd (Class I RNA is ³⁰ n M and Class II RNA is ⁶⁰ n M,) (this concentration is excessive 10 times 5 of thrombin), clotting time 1. There was a 5-fold increase (FIG. 34B).

Specificity of thrombin binding . Representative ligands from class I and class II showed that these ligands have low affinity for AT III even at high concentrations of 1 μM. These ligands showed low affinity compared to large amounts of 30N3 RNA, suggesting that there was a selection for non-specific binding. This is particularly important because AT III is a large amount of plasma protein that exhibits high affinity for the polyanionic polymer heparin. These results indicate that the distinct structural evolution present in class I and class II RNAs is specific for thrombin binding and binds to high affinity heparin binding proteins despite its polyanionic composition. Indicates not to. It is also noted that these thrombin specific RNA ligands are inactive biochemical precursors of active thrombin and have no affinity for prothrombin circulating at high levels (about 1 μM) in plasma. Should be (FIG. 35B).

Nucleic acid ligand for basic fibroblast growth factor (bFGF) . The present invention applies the SELEX method to specific target bFGF. The experimental section below lists the experimental parameters used to isolate and identify nucleic acid ligand solutions for bFGF.

  The present invention includes specific nucleic acid ligands shown in Tables II-IV. The range of ligands covered by the present invention extends to all ligands for bFGF identified by the SELEX method. More specifically, the present invention includes nucleic acid sequences that are substantially opposite to the specific nucleic acid ligands shown in Tables II-IV and have substantially the same ability to bind bFGF.

  For the family 1 and 2 ligands, the proposed structure formation summary shown in FIG. 41 shows that sequences with little or no homology to the primary sequence still have substantially the same ability to bind to bFGF. It shows that. The present invention also includes RNA ligands having substantially the same structure as the ligands provided herein and having substantially the same ability to bind bFGF to the RNA ligands shown in Tables II and III. . “Substantially identical structure” includes all RNA ligands having common elements of the sequences shown in Tables II and III (SEQ ID NOs: 27-67).

The present invention also includes the above-described ligands with certain chemical modifications to increase the in vivo stability of the ligand, facilitate or mediate delivery of the ligand in the body, or reduce the clearance rate from the body. In particular, RNA ligands of bFGF containing 2′-NH ₂ modifications of ribose with RNA ligands are within the scope of the present invention.

  The nucleic acid ligands and nucleic acid ligand solutions for bFGF described herein are useful as drugs and as part of gene therapy. Furthermore, the nucleic acid ligands for bFGF described herein can be advantageously used for diagnostic purposes.

  The high affinity nucleic acid ligands of the present invention also have various properties, including the ability to inhibit the biological activity of bFGF. Representative ligands from sequence families 1 and 2 were found to inhibit the binding of bFGF to both low- and high-affinity cell surface receptors. These nucleic acid ligands can be useful as specific and potent neutralizing agents for bFGF activity in vivo.

Example I: Improved nucleic acid ligand solution elucidation for HIV-RT RNA synthesis . In vitro transcription with oligonucleotide templates was performed as described in Milligan et al. (1987) (supra). All synthetic nucleic acids were made using an Applied Biosystems model 394-08 DNA / RNA synthesizer (Standard Bioprotocol model 394-08 DNA / RNA synthesizer) using standard protocols. Deoxyribonucleotide phosphoramidites and solvents and reagents for DNA synthesis were purchased from Applied Biosystems. Ribonucleotides and 2'-methoxy-ribonucleotide phosphoramidites were purchased from Glen Research Corporation. For mixed base positions, 0.1 M bosphoramidite solution was mixed in the indicated volume ratio. Base deprotection was performed in ammonium hydroxide: ethanol 3: 1 at 55 ° C. for 6 hours. The t-butyl-dimethylsilyl protecting group was removed from the 2′-OH group of the synthetic RNA by treatment in tetrabutylammonium fluoride overnight. The deprotected RNA was then phenol extracted, ethanol precipitated and purified by gel electrophoresis.

Affinity measurement using labeled RNA and HIV-RT . Model RNA for the improvement of 5 'and 3' boundaries and measurement of the effect of substitution was performed in a reaction of 0.5 mM C, G, and UTP with 0.05 mM ATP during transcription with T7 RNA polymerase. , Except that α- ³² P-ATP was used, as described by Turk & Gold (1990) (supra). Synthetic oligonucleotides and phosphorylated transcripts (Tuerk & Gold (1990) (supra) are kinases as described by Gauss et al. (1987) Mol. Gen. Genet. 206 : 24. All RNA-protein binding reactions were performed in a “binding buffer” consisting of 200 mM KOAc, 50 mM Tris-HCl pH 7.7, 10 mM dithiothreitol, with the exception of the chemoprotection experiments noted below. Dilutions of RNA and protein were mixed and stored on ice for 30 minutes and then transferred for 5 minutes to 37 ° C. For binding measurements, the reaction volume was 60 μl, of which 50 μl was measured. 3 ml of binding buffer by aspiration through a pre-moistened nitrocellulose filter (with binding buffer) Rinse and then dry and count for measurement or elute and measure for chemical modification.For binding affinity comparison, plot the results and use the graph to determine the protein concentration at which half maximum binding occurs. (Approx. Kd under protein-excess conditions) was determined.

Selection of modified RNA by HIV-RT . The binding reaction was as described above, except that the volume of the reactant was increased to a lower concentration rather than changing the amount of HIV-RT added to the reactant. RNA modified under denaturing conditions was selected with 20, 4 and 0.8 nanomolar concentrations of HIV-RT (1, 5 and 25 ml in binding buffer volume). The amount of RNA added to each reaction was equivalent for each experiment (about 1-5 pmol). RNA was eluted from the filter as described in Turk & Gold (1990) (supra) and measured for modified positions. Each experiment included a control in which unselected RNA was spotted on the filter in parallel with the selected RNA and eluted and measured for modified positions. Changes in the chemical modification of the selected RNA and unselected RNA were made by visually examining the exposed film of the electrophoresed measurement product with the following exceptions. The degree of modification interference by ENU was measured by scanning the density of the film using an LKB laser densitometer. The index of modification interference (M.I.) at each position was calculated as follows:
M.M. I. = (OD unselected / OD unselected A20) / (OD selected / OD selected A20)
Where the value of each position measured for the selected modified RNA (OD selection) is divided by the value for position A20 (OD selection A20) and similarly divided for the unselected lanes. Standardized values were used. All M.O. 1. Values are reported as disturbing, and 4.0 and higher are reported as strongly disturbing. In measuring the effect of mixed displacement of 2′-hydroxyl (on the ribose of each nucleotide) with 2′-methoxy, the gel of the electrolyzed hydrolyzed product was directly measured with an Ambis detection system. The count associated with each band in the lane was normalized as described above for position A17. Furthermore, it measured with the laser densitometer as described below.

Chemical modification of RNA . There is a useful review of the types of chemical modification of RNA and its specificity and methods of measurement by Ehresmann et al. (1987) (supra). Modification of RNA under native conditions was performed with 200 mM KOAc, 50 mM Tris-HCl pH 7.7 at 37 ° C. with ethyl nitrosourea (ENU) (1/5 v / v dilution of ENU saturated ethanol at room temperature). 1-3 hours, 8 minutes with dimethyl sulfate (DMS) (1/750 fold v / v dilution), 8 minutes with ketoxal (0.5 mg / ml), 20 minutes with carbodiimide (CMCT) (8 mg / ml), Then, for 45 minutes with diethylpyrocarbonate (DEPC) (1/10 v / v dilution under native conditions or 1/100 dilution under denaturation conditions), add 1 mM DTT under the same conditions. To HIV-RT. The concentration of the modifying chemical reagent was the same under denaturing conditions (except for the portion described for DEPC); these conditions were 7M urea except when modified with ENU in the absence of 7M urea. 50 mM Tris-HCl pH 7.7, 1 mM EDTA at 90 ° C. for 1-5 minutes.

Measurement of chemical modification . Elongated Ligand B RNA, 5'-GGUCCGAAGU25GCAACGGGGAAAUGCACUAUUGAAGAAU-UUUAUAUCUCUAUUGUAAC-3 '
The position of chemical modification was determined by reverse transcription for DMS, ketoxal and CMCT on (SEQ ID NO: 11) (ligand B sequence is underlined); here oligonucleotide primer 5′-CCGGATCCGTTTCAATAGAG-ATATAAAATTC-3 '(SEQ ID NO: 12) was annealed; reverse transcripts (obtained as previously described by Gauss et al., 1987) were separated by electrophoresis on a 10% polyacrylamide gel. The positions of the ENU and DEPC modifications are described in Vlassov et al. (1980) FEBS Lett. 120 : 12, and Pettie and Gilbert (1980) Proc. Natl. Acad. Sci. USA 77 : 4679 (separated by electrophoresis on a 20% acrylamide gel). Measurement of 2'-methoxyribose for ribose at various positions was measured by alkaline hydrolysis in 50 mM sodium carbonate (pH 9.0) at 90 ° C for 45 minutes.

Modification of RNA in the presence of HIV-RT . The conditions used were the same as for the modification of native RNA. The concentration of HIV-RT was approximately 10-fold excess of the RNA concentration. In general, protein concentrations ranged from 50 nM to 1 μM.

SELEX isolation of incidental contact with HIV-RT . The starting RNA was transcribed from a PCR-reacted template synthesized from the following oligonucleotides:

(5 'primer) (SEQ ID NO: 13),

(3 'primer) (SEQ ID NO: 14),

(Variable template) (SEQ ID NO: 15).
SELEX was performed as previously described for HIV-RT with the following exceptions. The concentration of HIV-RT for the first SELEX binding reaction was 13 nmol in 4 ml binding buffer, 10 μmol RNA, and 2.6 nmol HIV-RT in 20 ml buffer for the second through ninth time. , 1.8 micromolar RNA was selected, 10-14th using 1 nanomolar HIV-RT in 50 ml, 0.7 micromolar RNA, and 15th and 16th in 50 ml binding buffer. 0.5 nanomolar HIV-RT, 0.7 micromolar RNA was used.

  References for Example 1:

Example II: Evaluation of Improved Nucleic Acid Ligand Solution for HIV-1 Rev Protein The Rev ligand sequence used for chemical modification is shown in FIG. 12 (hereinafter numbered schematics are used). RNA for modification was obtained from T7 RNA polymerase transcription of a synthetic oligonucleotide template. ENU modification was performed on the ligand sequence shown in FIG. DMS, ketoxal, CMCT, and DEPC modifications were performed on the extended ligand sequence and analyzed by reverse transcription using the synthetic oligonucleotide primers shown in FIG.

Chemical modification of RNA . Nucleic acid chemical modification techniques are generally described in Ehresmann 1 et al. (1987) (supra). Modification of RNA under native conditions was performed at 37 ° C. in 200 mM KOAc, 50 mM Tris-HCl pH 7.7, 1 mM EDTA. Modification under denaturing conditions was performed at 90 ° C. in 7 M urea, 50 mM Tris-HCl pH 7.7. The concentration of the modifying reagent and the incubation time are as follows: Ethyl nitrosourea (ENU)-1/5 v / v dilution of ENU saturated ethanol, unmodified 1-3 hours, denatured 5 minutes; dimethyl sulfate (DMS)- 1 / 750-fold v / v dilution, undenatured 8 minutes, denatured 1 minute; ketoxal-0.5 mg / ml, undenatured 5 minutes, denatured 2 minutes; carbodiimide (CMCT) -10 mg / ml, undenatured 30 minutes, Denaturation 3 minutes; v / v dilution of diethyl pyrocarbonate (DEPC) -1/10, unmodified 10 minutes, modified 1 minute.

Interfering with Rev binding modification . RNA chemically modified under denaturing conditions was selected for Rev binding by filter fractionation. Selection was performed at Rev concentrations of 30, 6, and 1.2 nmoles (binding buffer (200 mM KOAc, 50 mM Tris-HCl pH 7.7, and 10 mM dithiothreitol) in volumes 1, 5, and 25 ml. Equivalent to Approximately 3 pmoles of modified RNA was added to each protein solution, mixed and stored on ice for 15 minutes, then transferred to 37 ° C. for 10 minutes. The binding solution was passed through a pre-moistened nitrocellulose filter and rinsed with 5 ml binding buffer. RNA was eluted from the filter as described by Turk & Gold (1990) (supra) and measured for the remaining modified positions. The modified RNA was also spotted on a filter and eluted to elute for uniform recovery of the modified RNA.

  The degree of modification interference was measured by autoradiographic density scanning using LKB (ENU) and molecular dynamics (DMS, ketoxal, CMCT, and DEPC) laser densitometers. Values for modified phosphate and base were normalized at the selected modification positions for both selected and unselected lanes; values for modification positions of the next selected lane correspond to the unselected lanes. (See Figures 15-19 for specific standardized positions). A value of 4.0 or higher for the modified base and phosphoric acid was determined to be strongly interfering, and a value of 2.0 or higher was determined to be slightly interfering.

Modification of RNA in the presence of Rev. Rev ligand “footprints”, which are modifications of RNA ligands in the presence of Rev protein, were performed in 200 mM KOAc, 50 mM Tris-Cl pH 7.7, 1 mM DTT, and 5 mM MgCl ₂ . The protein concentration was 500 nanomolar and an approximately 3-fold molar excess of the RNA concentration. Modifications in the presence of protein were attempted using all the above-described modification reagents except ethylnitrosourea (ENU).

Measurement of chemically modified RNA . The position of the ENU modification was detected as in Vlassov et al. (1980) (described above) and separated by electrophoresis on a 20% denaturing acrylamide gel. DMS, ketoxal, CMCT, and DEPC were measured by reverse transcription of the extended Rev ligand with radiolabeled oligonucleotide primers (Figure 12) and separated by electrophoresis on an 8% denaturing acrylamide gel.

Cellex by biased randomization. Templates for in vitro transcription were prepared by PCR from the following oligonucleotides:

(Template Oligo) (SEQ ID NO: 16)

(5 'primer) (SEQ ID NO: 17)

(3 ′ primer) (SEQ ID NO: 18)
Here, the lower case letters of the template oligos indicate that a 62.5% amount of lower case letters at each position and a mixture of 12.5% each of the other three nucleotide reagents were used in the synthesis.

  SELEX performed as described above with the following exceptions. The concentration of HIV-1 Rev protein in the first and second binding reactions is 7.2 nmoles in a volume of 10 ml (200 mM potassium acetate, 50 mM Tris-HCl pH 7.7, 10 mM DTT) and RNA is 4 micromolar. From the 3rd to the 6th round, in a volume of 40 ml, the Rev protein concentration was 1 nanomolar and the RNA was 1 micromolar. HIV-1 Rev protein was purchased from American Biotechnologies, Inc.

Example III: Nucleic acid ligand for HIV-1 tat protein .
SELEX of HIV-1 tat protein. The tat protein was purchased from American Bio-Technologies, Inc. Templates for in vitro transcription were prepared by PCR using the following oligonucleotides:

(5 'primer) (SEQ ID NO: 18)

(Variable template) (SEQ ID NO: 19)

(3 ′ primer) (SEQ ID NO: 20)
The SELEX rotation was performed as described in Turk et al. (1992a) (supra) and the SELEX application under the following conditions: The binding reaction was performed at 13 nmoles in a volume of 2 ml for 1 and 52 rounds. The test was carried out with tat protein and 1.3 micromolar RNA, and the 3rd round was performed with 6.5 nanomolar tat protein and 0.65 micromolar RNA in 4 ml.

RNA synthesis . In vitro transcription with oligonucleotide templates was performed as described by Milligan et al. (1987) (supra). All synthetic nucleic acids were made on an Applied Biosystems model 394-08 DNA / RNA synthesizer using standard protocols. (Applied Biosystems model 394-08 DNA / RNA synthesizer) Deoxyribonucleotide phosphoramidites and solvents and reagents for DNA synthesis were purchased from Applied Biosystems.

Affinity measurement between labeled RNA and HIV-1 tat protein . Model RNA for improving the 5 'and 3' boundaries and measuring the effect of substitution was used to react 0.5 mM C, G, and UTP with 0.05 mM ATP during transcription with T7 RNA polymerase. Labeled as described in the SELEX application except that α- ³² P-ATP was used. All RNA-protein binding reactions were performed in a “binding buffer” consisting of 200 mM KOAc, 50 mM Tris-HCl pH 7.7, 10 mM dithiothreitol. The RNA and protein dilutions were mixed and stored on ice for 30 minutes and then transferred to 37 ° C. for 5 minutes. In the binding measurement, the reaction volume was 60 μl, of which 50 μl was measured. Each reaction is aspirated through a pre-moistened (with binding buffer) nitrocellulose filter, rinsed with 3 ml binding buffer, then dried and counted for measurement or eluted and measured for chemical modification. did. For binding affinity comparison, the results were plotted and the protein concentration at which half-maximal binding occurred (approximately Kd under protein-excess conditions) was determined from the graph. The result of the binding measurement is shown in FIG.

Example IV: Nucleic Acid Ligand for Thrombin A high affinity RNA ligand for thrombin was isolated by SELEX. The random RNA molecules used in the initial candidate mixture were generated by in vitro transcription from a 102 nucleotide double stranded DNA template containing a random cassette 30 nucleotides (30N) in length. A population of 10 ¹³ 30N DNA templates were generated by PCR using a T7 promoter for in vitro transcription and a 5 'primer containing restriction sites in both the 5' and 3 'primers for cloning. .

The concentration of RNA at each rotation of SELEX is about 2-4 × 10 ⁻⁷ M, and the concentration of thrombin (Sigma, 1000 units) from the first 1.0 × 10 ⁻⁶ to 2 and 3 The fourth time was 4.8 × 10 ⁻⁷ , and the fourth time was 12 × 2.4 × 10 ⁻⁷ . The RNA and protein binding buffer was 100 mM NaCl, 50 mM Tris / Cl, pH 7.7, 1 mM DTT, and 1 mM MgC positive2. Binding was performed at 37 ° C. for 5 minutes in a total volume of 100 μl for the 1-7th and 200 μl for the 8-12th. Each binding reaction was filtered through a pre-wet (50 mM Tris / Cl, pH 7.7) nitrocellulose filter (2.5 cm millipore, 0.45 μM) in a Millipore filter binding device and immediately 5 ml Rinse with the same buffer. The RNA was removed from the filter with 400 μl phenol (equilibrated with 0.1 M NaoAc pH 5.2), as described (Tuerk et al. (1990) supra), with 200 μl freshly prepared 7 M urea. Eluted. The RNA was precipitated with 20 μg of tRNA and used as a template for cDNA synthesis, followed by PCR and transcribed in vitro to prepare the next round of RNA. On the 1st to the 8th round, RNA was radiolabeled with ³² P-ATP so that binding could be monitored. In order to speed up the time of each SELEX, RNA was not labeled at 9-12. RNA was pre-filtered with a nitrocellulose filter (1.3 cm millipore, 0.45 μM) prior to 3, 4, 5, 8, 11, and 12 to exclude selection for non-specific nitrocellulose binding. Filtered.

Binding curves were drawn after the 5th, 8th and 12th rounds in order to estimate the kD change of large amounts of RNA. These experiments were performed at a protein excess of 1.2 × 10 ⁻⁵ to 2.4 × 10 ⁻⁹ M at a final RNA concentration of 2 × 10 ⁻⁹ M. The RNA for these binding curves was labeled for high specific activity with ³² P-ATP or ³² P-UTP. Binding to the nitrocellulose filter was as described for each SELEX except that the RNA bound to the filter was dried and measured directly on the filter.

RNA sequencing . Following the 12th SELEX, RNA was sequenced with reverse transcriptase (AMV, Life Sciences, Inc.) using ³² P5 ′ end-labeled 3 ′ complementary PCR primers.

Cloning and sequencing of individual RNAs . RNA from the 12th round was reverse transcribed into DNA and amplified by PCR. Digested with restriction enzyme sites in the 5 ′ and 3 ′ fixed regions were used to remove the 30N region, which was then ligated to the complementary sites of the E. coli cloning vector pUC18. The ligated plasmid DNA was transformed into JM103 cells and selected by blue / white colony formation. Colonies containing unique sequences were grown and miniprep DNA was prepared. Double-stranded plasmid DNA was used for dideoxy sequencing using Sequenase kit version 2.0 (Sequenase kit version 2.0) and ³⁵ S-dATP (Amersham).

End-labeled RNA . For end labeling, RNA transcribed with T7 polymerase was gel purified by UV shielding. The 5 ′ end of the RNA was labeled by dephosphorylating the 5 ′ end with 1 unit alkaline phosphatase for 30 minutes at 37 ° C. Alkaline phosphatase activity was destroyed by phenol: chloroform extraction. The RNA was then end-labeled with γ ³² P-ATP by reaction with polynucleotide kinase at 37 ° C. for 30 minutes.

The RNA, with 30 minutes (5'- ³² P) pCp and RNA ligase at 37 ° C., was labeled 3 'end. The 5 ′ and 3 ′ end-labeled RNAs were gel band purified on 8 & 8M urea polyacrylamide gels.

Determination of 5 'and 3' boundaries . Two picomoles of RNA 3 ′ or 5 ′ end-labeled for 5 ′ or 3 ′ boundary experiments, respectively, were dissolved in 50 mM Na ₂ CO ₃ (pH 9.0) and 1 mM EDTA in 10 μl reaction. Hydrolyzed for 10 minutes at ° C. The reaction was stopped by adding 1/5 volume of 3M NaOAc (pH 5.2) and frozen at -20 ° C. Three protein concentrations (40 nM, 10 nM and 2.5 nM) in three volumes (100 μl, 400 μl, and 1600 μl, keeping the amount of protein constant) containing 1 × binding buffer and 2 picomoles of RNA ) To carry out a binding reaction. The reaction was incubated at 37 ° C. for 10 minutes, filtered through a pre-moistened nitrocellulose membrane and rinsed with 5 ml wash buffer. The filter was cleaved and RNA was eluted from the filter by shaking it in 200 μl 7M urea and 400 μl phenol (pH 8.0) at 20 ° C. for 15 minutes. After adding 1200 μl of H ₂ O, the phases were separated and the aqueous phase was extracted once with chloroform. RNA was precipitated with 1/5 volume of 3M NaOAc, 20 μg carrier tRNA, and 2.5 volumes of ethanol. The pellet was washed once with 70% ethanol, dried and resuspended in 5 μl H20 and 5 μl formamide developing dye. The remainder of the alkaline hydrolysis reaction was diluted 1:10 and an equal volume of developing dye was added. To find where the boundaries were on the sequence ladder (1adder), the RNase T1 digest of the ligand was electrophoresed in parallel with the alkaline hydrolysis and binding reactions. This digestion was performed in a 10 μl reaction containing 500 units of end-labeled RNA in 7 M urea, 20 mM sodium citrate (pH 5.0) and 1 mM EDTA and 10 units of RNase T1. The RNA was incubated for 10 minutes at 50 ° C. without enzyme, then incubated for an additional 10 minutes after addition of enzyme. The reaction was slowed by adding 10 μl of developing dye and incubating at 4 ° C. Immediately after digestion, 5 μl of each digest, hydrolyzate, and three binding reactions were electrophoresed on a 12% sequencing gel.

In vitro transcription of 2-NH2 ribose derivatives of UTP and CTP RNA . RNA was transcribed directly from the pUC18 plasmid miniprep dsDNA template using T7 RNA polymerase in a reaction containing ATP, GTP, 2-NH ₂ -UTP and 2-NH ₂ -CTP. For ³² P- labeled ^RNA, including 32 P-ATP in the reaction. Unmodified RNA was transcribed in a mixture containing ATP, GTP, UTP, and CTP.

  RNA synthesis. RNA molecules corresponding to the lower limit of the nucleotide sequence required for high affinity binding to thrombin, as determined by boundary experiments (Figure 30B), were applied to an Applied Biosystems 394 DNA / RNA synthesizer (Applied Biosystems 394 DNA / RNA synthesizer). Was synthesized. These RNA molecules comprise a 24 nucleotide (24R) and 39 nucleotide (39R) class I clone 16 hairpin structure and a 33 nucleotide (33R) class II clone 27 hairpin.

Binding of individual RNA molecules . Four DNA plasmids with unique 30N sequences were selected for in vitro transcription. ³² P-labeled RNA was transcribed with conventional nucleotides and 2-NH ₂ derivatives of CTP and UTP. The binding curves for these individual RNAs were established using binding buffer and concentrations of 1.0 × 10 ⁻⁵ to 1.0 × 10 ⁻¹⁰ M thrombin (1000 units, Sigma). Human α thrombin (Enzyme Research Laboratories, ERL) is also used to measure RNA binding affinity at concentrations from 1.0 × 10 ⁻⁶ to 1.0 × 10 ⁻¹⁰ M. The binding of 5 ′ end-labeled single strand 15: mer DNA 5′-GGTTGGTGTGGTTGG-3 ′ (G15D) (SEQ ID NO: 1) described by Bock et al. It was measured with ERL thrombin under the binding conditions described in the specification and compared with binding by the radiolabeled RNA hairpin structure described above.

Competition experiments. In order to detect whether the described RNA ligands can compete for the binding of DNA 15-mer G15D to thrombin, equimolar concentrations (1 μM) of thrombin and 5′-end labeled DNA 15-mer G15D were used at a concentration of 10 nM from the presence and in the absence of a "cold" unlabeled RNA or DNA ligand was changed to 1 [mu] M, were incubated with a filter binding conditions (kD of approximately 200 n ^M). In the absence of competition, RNA binding was 30%. Finally, protein was added so that competition for binding occurred. The RNA ligands tested for competition were class I clone 16 synthetic RNA 24-mer (24R) and 39-mer hairpin (39R), and class II 27 synthetic RNA 33-mer (33R). Results are expressed as the relative fraction of bound G15D (G15 with competitor / G15 without competitor) versus the concentration of cold competitor.

For Class I RNA to determine whether to compete for binding with class II RNA, and to confirm the competition with G15DDNA, thrombin and 5 'end-labeled RNA33 hairpin Class II equimolar ⁽³⁰⁰ n M) the "cold" was changed from 100 ⁿ M to 32μM concentration in the presence or absence of unlabeled RNA24 or DNA G15D, and incubated with a filter binding conditions. Results are expressed as the relative fraction of bound RNA33 (RNA33 in the presence of competitor / RNA33 in the absence of competitor) versus cold competitor concentration (FIG. 33).

Colorimetric measurement of thrombin activity and inhibition by RNA ligands . Hydrolysis of the chromogenic substrate S-2238 (HD-Phe-Pip-Arg-p nitroaniline [HD-Phe-Pib-Arg-pNA]) (mold-Pharmacia) by thrombin, p from the substrate. -Measured optically at 405 nm by release of nitroaniline (pNA).

Thrombin _H-D-Phe-Pip- Arg-pNA + H 2 O -------->
HD-Phe-Pip-Arg-OH + pNA

Thrombin in a reaction buffer (50 mM sodium citrate, pH 6.5, 150 mM NaCl, 0.1% PEG) containing 250 μM S2238 substrate to a final concentration of 10 ⁻⁸ or 10 ⁻⁹ M. Added at 37 ° C. For inhibition measurements, thrombin and RNA (in equimolar or 10-fold excess) were preincubated for 30 seconds at 37 ° C. (FIG. 34A) before being added to the reaction mixture.

Fibrinogen coagulation. Contains 0.25 mg / ml fibrinogen and 1 u / λ RNAse inhibitor (RNAasin, Promega) with or without 30 nM RNA class I or 60 nM RNA class II at 37 ° C. , 400 μl of incubation buffer (20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ ) with thrombin to a final concentration of 2.5 nM Added. The time from thrombin addition to coagulation formation was measured in seconds by a tilt test (FIG. 34B).

Specificity of thrombin binding. The binding affinity of full-length class I RNA16, class II RNA27, and large amounts of 30N3 RNA to the serum proteins antithrombin III (AT III) and prothrombin was reduced by filter binding as described above for the evolution of high affinity RNA ligands. It was measured. These experiments were performed at a final RNA concentration of 2 × 10 ⁻⁹ M with a protein excess of 1 × 10 ⁻⁵ to 5 × 10 ⁻¹⁰ M (FIG. 35).

Example V: Nucleic acid ligand for bFGF .
Material . bFGF was obtained from Bachem California (molecular weight 18,000 daltons, 154 amino acids). Tissue culture grade heparin (average molecular weight 16,000 daltons) was purchased from Sigma. Low molecular weight heparin (5,000 daltons) was obtained from Calbiochem. All other chemicals were purchased at least reagent grade and commercially available.

SELEX . The basic features of the SELEX protocol are described in detail in the aforementioned report (Turck & Gold (1990) (previously); Turk et al (1992a) (previously); Turk et al (1992b) polymerase chain. Reaction (Polymerase Chain Reaction) (Ferre, F., Muris, K., Gibbs, R. and Ross, A. (Edited by Ferre, F. Mullis, K. Gibbs, R. & Ross, A.)) Berkhauser (Birkhauser), New York). Briefly, a DNA template for in vitro transcription (which contains a region of 30 random positions adjacent to the constant sequence region) and the corresponding PCR primer were chemically synthesized (operon). Random regions were created by using an equimolar mixture of 4 nucleotides during oligonucleotide synthesis. The two constant regions were designed to contain a PCR primer annealing site, a primer annealing site for cDNA synthesis, a T7 RNA polymerase promoter region, and a restriction enzyme site that allows cloning into vectori (Table 1). See I).

The first pool of RNA molecules, using T7 RNA polymerase (New England Biolabs (New England Biolabs), prepared by in vitro transcription of double-stranded DNA template about 200 picomoles (pmol) ^{(10 14} molecules) The transcription mixture consisted of 100-300 nM template, 5 units / μl T7 RNA polymerase, 40 mM Tris-C1 buffer (pH 8.0) containing ₁₂ mM MgC12, 5 mM DTT, 1 mM spermidine, 0 Composed of 0.002% Triton X-100 and 4% PEG The transcription mixture was incubated for 2-3 hours at 37 ° C. These conditions typically yielded 10 to 100-fold transcription amplification. It was.

Selection for high affinity RNA ligands was 50 μl phosphate buffered saline (PBS) (10.1 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , 137 mM NaCl, 2.7 mM In KCl, pH 7.4), bFGF (10-100 pmol) was incubated with RNA (90-300 pmol) at 37 ° C. for 10 minutes, and protein-RNA complexes were separated from unbound species by nitrocellulose filter fractionation. (Turk & Gold (1990), supra). The selected RNA (which typically amounts to 0.3-8% of the total input RNA) is then extracted from the filter and avian myeloblastosis virus reverse transcriptase (AMVRT, Life Sciences) was reverse transcribed into cDNA. Reverse transcription was performed at 50 ° C. (30 minutes) in 50 mM Tris buffer (pH 8.3), 60 mM NaCl, 6 mM Mg (OAc) 2, 10 mM DTT, and 1 unit / μl AMV RT. . Amplification of cDNA by PCR under standard conditions yielded sufficient quantities of double stranded DNA for the next round of in vitro transcription.

Nitrocellulose filter binding measurement . Oligonucleotides bound to proteins, effectively by filtration through nitrocellulose membrane filters, it is separated from unbound species (Yarusu and Berg (Yarus & Berg) (1970) Anal.Biochem 35:. 450; Rowari and Uhlenbeck ( Lowry & Uhlenbeck (19S7) Nucleic Acids Res. 15 : 10483; Turk & Gold (1990) (previously described) Nitrocellulose filter (Millipore, 0.45 μm pore size, HA type) filter manifold And washed with 4-10 ml buffer, ³² P-labeled RNA with serial dilutions of protein in buffer (PBS) containing 0.01% human serum albumin (HSA) at 37 ° C. At After cubating (5-10 minutes), the solution was applied to the filter in 45 μl portions under low vacuum and washed with 5 ml of PBS, which was then dried under an infrared lamp and counted on a scintillation counter. Cloning and sequencing Individual members of the enriched pool were cloned into the pUC18 vector and sequenced as described (Schneider et al. (1992) (supra); & Gold) (1990) supra).

SELEX experiment targeting bFGF . Following the above steps, one SELEX experiment (experiments A and B) targeting bFGF was initiated using separate pools of randomized RNA (each pool consisting of approximately 10 ¹⁴ molecules). The constant sequence region adjacent to the randomized region, with the corresponding primer, was different in each experiment. One template / primer combination used is shown in Table I.

  Selection was performed at 37 ° C in PBS. The selection performed in Experiment B was performed in the presence of heparin (Sigma, molecular weight 5,000-32,000 daltons, average molecular weight 16,000 daltons) in the molar ratio 1/100 (heparin / bFGF) in the selection buffer. Performed in liquid. Heparin competes for binding of randomized RNA to bFGF and the amount of heparin. The amount of heparin used significantly reduced RNA binding to bFGF, but did not eliminate it (data not shown). There was one basis for using heparin. First, heparin is known to induce small changes in protein configuration and stabilize bFGF against temperature denaturation. Second, the apparently competing nature of heparin binding to randomized RNA to bFGF increases the stringency of selection for the heparin binding site or directs RNA ligand binding to another site. Was expected.

  A significant improvement in the affinity of the RNA ligand for bFGF was observed after 10 rotations in experiment A and 13 rotations in experiment B. Sequencing these enriched pools of RNA ligand revealed a clear departure from randomness, indicating that the number of molecules remaining in the pool was substantially reduced. Individual members of the enriched pool were then cloned into the pUC18 vector and sequenced as described above.

49 clones from Example A and 37 clones from Example B were sequenced. Of the 86 sequences in total, 71 sequences were unique. One distinct family was identified based on overlapping regions of sequence homology (Tables II and III). As expected (Irvine et al. (1991) J. Mol. Biol. 222 : 739), there are many sequences that have no apparent homology to any member of a family. Shown in Table IV.

  The consensus sequence for Family 1 ligands (Table II) is defined by a contiguous range of 9 bases (CUAACCAGGG (SEQ ID NO: 27)). This suggests a minimal structure consisting of a 4-5 nucleotide loop containing a strongly conserved AACC sequence and a raised axis (Figure 41 and Table VI). The Family 2 ligand consensus sequence (Table III) is further extended and contains a less conserved region, RRGGHAACGGYWNGDCAAAGNCCYY (SEQ ID NO: 43). Here, most strongly conserved positions are present in large (19-21 nucleotide) loops (FIG. 41 and Table III). Additional structures within the loop are possible.

  The presence of one distinct sequence family of RNA enriched pools suggests that there is one convergent solution for high affinity binding to bFGF. SELEX experiment A related members to both sequences (Table II). On the other hand, all sequences from SELEX experiment B (selected in the presence of heparin) belong to Family 2 (Table III) or “Other Sequences” family (Table IV) but are not found in Family 1 It was. This is surprising in view of the fact that bFGF was present in a 100-fold molar excess of heparin during selection. However, the effective molar excess of bFGF over heparin was probably much smaller. The average molecular weight of heparin used for selection was 16,000 daltons. Each saccharide unit weighs 320 daltons, and since at least 8 saccharide units are required for high affinity binding to bFGF, an average of 6 bFGF molecules can bind to a heparin molecule. This reduces the ratio of heparin to bFGF to 1:16. Indeed, this amount of heparin is sufficient to reduce the observed affinity of the unselected RNA pool for bFGF by a factor of 5 (data not shown). The observed elimination of the entire ligand family due to the presence of a relatively small amount of heparin in the selection buffer may be the result of a change in protein configuration induced by heparin. Due to the relative amounts of heparin and bFGF used in the selection, this model is long enough for the heparin-induced configuration to persist after dissociation of the protein-heparin complex and the lifetime of this configuration is sufficient to equilibrate with the RNA ligand. Need to be.

  Family 2 sequences consist of clones derived from both SELEX experiments. This suggests that the adjacent constant region typically plays a relatively insignificant role when measuring the affinity of these ligands, and this family of consensus sequences has a high affinity for bFGF. Supports the premise that it is the main determinant of the bond.

Measurement of binding affinity for bFGF .
Equilibrium dissociation constant . In the simplest case, the equilibrium binding of RNA to bFGF is described by Equation 1:
RNA / bFGF ← → RNA + bFGF (1)
The fraction of RNA bound (q) is proportional to the concentration of free protein [P] (Equation 2)
q = f [P] / ([P] + Kd) (2)
Where Kd is the equilibrium dissociation constant and f reflects the efficiency of retention of the protein-RNA complex on the nitrocellulose filter. The average value of f for bFGF was 0.82.

  To exclude higher order structures, all RNA solutions were heated in PBS at 90 ° C. for 2-3 minutes and cooled on ice prior to protein incubation. After this treatment, only a single band of all RNA clones was detected on a non-denaturing polyacrylamide gel.

  The relative binding affinity of individual ligands for bFGF cannot be predicted from the sequence information. Thus, after incubation with 4 and 40 nM protein, unique sequence clones were selected for their ability to bind bFGF by measuring the fraction of radiolabeled RNA bound to the nitrocellulose filter. This screening method was accurate enough to identify a small number of clones with dissociation constants in the nanomolar range. The binding of these selected clones was then analyzed in more detail.

  High affinity RNA ligands for bFGF were found in both sequence families (Tables VI and VII). Clones that did not belong to either family generally had low affinity (data not shown).

  The original unselected RNA pool bound to bFGF with an affinity of 300 nM (set A) and 560 nM (set B) (FIG. 36). SELEX thus allowed the isolation of ligands with at least two orders of magnitude higher affinity for bFGF.

  To address specificity issues, a representative set of high affinity ligands for bFGF (family 1 to 5A and 7A; family 2 to 12A and 26A) were tested for binding to the other four heparin binding proteins. did. The affinity of these ligands for acidic FGF, thrombin, antithrombin III, and vascular endothelial growth factor was found to be relatively weak (Kd> 0.3 μM) (data not shown).

RNA ligand inhibition of bFGF receptor binding . The same four high affinity RNA ligands were also tested for their ability to inhibit binding of bFGF to low and high affinity cell surface receptors.

Consider receptor binding . bFGF was labeled with ¹²⁵ I by the Iodo-Gen (Pierce method) described by Moscatelli (1987) (supra). Wash and then αMEM medium containing serial dilutions of 10 nm / ml ¹²⁵ I-bFGF, 0.1% HSA, 1 unit / ml RNasein, and high affinity RNA in PBS And incubated for 2 hours at 4 ° C. In another experiment, it was established that RNA was not significantly degraded under these conditions: The amount of ¹²⁵ I-bFGF bound to the low and high affinity receptor sites. Is measured as described by Moscatelli (1987) (supra). All four ligands competed for the low affinity receptor site, while unselected (random) RNA did not compete (Figure 37A), resulting in half substitution of bFGF from the low affinity receptor. The concentration of RNA required for ligand 5A, 7A and 26A was 5-20 nM and for ligand 12A was> 100 nM Half substitution from the high affinity site was for ligands 5A, 7A and 26A Was observed at> 1 μM for ligand 12A, and random RNA did not compete with high affinity receptors to replace bFGF from low and high affinity receptors. The observed difference in the concentration of RNA required is the difference in the affinity of one receptor class for bFGF (low affinity In 2-10nM for position, for the high affinity site it is expected to reflect the 10-100pM).

  Heparin competitively displaced RNA ligands from both sequence families (FIG. 38), but higher concentrations of heparin were required to replace family 2 members from bFGF.

The selective advantage obtained with the SELEX method is based on the affinity for bFGF. RNA ligands can in principle bind to any site on the protein, so it is important to test the activity of the ligand with an appropriate functional measurement. A suitable functional experiment for the selected high affinity ligand is a test of the ability to inhibit the binding of bFGF to the cell surface receptor, since bFGF thus exerts its biological activity. The fact that some representative high affinity RNA ligands inhibited binding of bFGF to both receptor classes (due to relative binding affinity) indicates that these ligands are at or near the receptor binding site. Suggests binding. This concept is further supported by the observation that heparin competes for binding of these ligands to bFGF. High affinity ligands from Family 1 and Family 2 bind to different sites on bFGF. The invention also includes covalently linking components from two families of ligands into a single, more potent inhibitor of bFGF.
Table I. Oligonucleotides used for SELEX experiments A and B.

Table II. Family 1 sequences of random regions from SELEX experiments A and B.

Table III. Family 2 sequences of random regions from SELEX experiments A and B.

Table IV. Other sequences of random regions from SELEX experiments A and B.

Table V. Repeated sequence of random regions from SELEX experiments A and B.

Table VI. Secondary structure and dissociation constant (K _d ) for a representative set of high affinity ligands from Family 1.

^a Strongly stored positions are shown in bold. The constant region nucleotides are shown in lower case.
Table VII. Secondary structure and dissociation constant for a typical set of high-affinity ligands from family 2 (K _d).

^a Strongly stored positions are shown in bold. The constant region nucleotides are shown in lower case.

FIG. 1 shows a consensus false knot from primary and secondary SELEX experiments describing a high affinity inhibitory ligand for HIV-1 reverse transcriptase (HIV-RT). The consensus secondary structure is a false knot; the 5 'helix (axis 1) of this false knot is conserved at the primary sequence level and the 3' helix, ie axis 2, is not conserved. X indicates the position of the non-conserved nucleotide; X-X 'indicates the preferred base pair. As shown here, 26 nucleotides are numbered. FIG. 2 shows the improvement of the 5 ′ information boundary. A series of model ligands were synthesized from template oligos using T7 RNA polymerase (Milligan et al. (1987) Nucl. Acid. Res. 15 : 8783). The complete ligand B is depicted in the upper left. On the right end, the variation of individual ligands A to E occurring in the area enclosed by the square is shown. The individual binding curves for these model ligands are shown in the graph. FIG. 3 shows the effect of various nucleotide substitutions within the ligand B sequence on binding to HIV-RT. Various substitutions and the resulting affinity for HIV-RT expressed as a percentage of ligand B binding are described. Ligand B is the control tested in each experiment; ligand B affinity is normalized to 1.0 to indicate relative affinity (Kd for ligand B divided by Kd for each ligand). Also shown is the affinity of ligand B with various ends truncated. The value associated with GG with an asterisk replacing U1-G16 is derived from ligand C in FIG. FIG. 4 shows a chemical probe in a non-denatured conformation versus a denatured conformation of ligand B. Various nucleotides of ligand B were reacted with chemicals under non-denaturing and denaturing conditions to determine the modified position, subjected to electrophoresis, and visualized and compared for comparison. The black square indicates the highly reactive base pairing group of the base at that position, the white square indicates partial reactivity; the black triangle indicates strong reactivity at the purine N7 position, and the white triangle indicates partial reactivity. (As opposed to modification with DEPC). The question mark indicates that these positions on G (-2) and G (-1) were indistinguishable due to the crowding of bands on the gel. FIG. 5 shows the reactivity of the modifiable group of ligand B when bound to HIV-RT. Groups that showed altered reactivity when bound to HIV-RT compared to the reactivity of the undenatured conformation are shown in the figure. FIG. 6 shows the results of modification interference of ligand B that forms a complex with HIV-RT. The symbols for modification are as in the legend enclosed in the square. Shows modifications that are strongly (black symbols) or partially (white symbols) selected for binding to HIV-RT (reflected by modifications that decrease at that position in the selected population). FIG. 7 shows the substitution of 2'-methoxy instead of hydroxyl on the ribose of the ligand B sequence shown in the upper right. White circles indicate hydroxyl groups at the indicated positions, and black circles indicate methoxy substitution. FIG. 8 shows selection by HIV-RT from a mixed population of 2′-methoxy ribose for 2′-hydroxyl at positions U1 to A5 and A12 to A20. The following sequence (sequence ID number: 2): 5 ′-(AAAAAA) _d (UCCGA) _x (AGUGCA) _m (ACGGGAAA) _x (UGCACU) _m-3 (where the subscript “d” is 2 ′) -Deoxy, subscript "x" indicates that these nucleotides are mixed 50-50 with phosphoramidite reagent to give 2'-methoxy or 2'-hydroxyl on ribose, and subscript The letter rm) synthesized an oligonucleotide with these nucleotides) indicating that all of these nucleotides are 2'-methoxy on ribose. FIG. 9 shows the starting RNA and a collection of sequences obtained from SELEX (SEQ ID NO: 115-135) using HIV-RT as part of the experiment. FIG. 10 shows the consensus extended HIV-RT ligand (SEQ ID NO: 136) obtained from the list of sequences shown in FIG. FIG. 11 shows a revised description of the HIV-RT false knot ligand. In addition to the promise for the label of FIG. 1, S-S 'indicates the preferred CG or GC base pair at this position. FIG. 12 shows the sequence of the high affinity RNA ligand of the HIV-1 Rev protein obtained from the SELEX experiment. A numbered schematic used for a particular base of this RNA is shown. This sequence was used for chemical modification with ENU. FIG. 12 also shows the extended RNA sequences used in a) for chemical modification experiments with DMS, ketoxal, CMCT, and DEPC in a). The sequence of the oligonucleotide used for the primary extension of the extended ligand sequence is shown in b). FIG. 13 shows the results of chemical modification of HIV-1 Rev ligand RNA under non-denaturing conditions. a) lists chemical modifying reagents, their specificity and symbols indicating partial and complete modification. The RNA sequence is indicated, along with the degree and type of modification displayed for each modified base. b) shows the helix, protuberance, and hapin structural elements of the HIV-1 Rev RNA ligand corresponding to the modification and computer structure prediction data. FIG. 14 shows the results of chemical modification of ligand RNA that interferes with binding to HIV-1 Rev protein. Modifications that interfere with protein binding were described and classified into strong and weak disturbances. The symbol means either base pairing modification, N7 modification, or phosphate modification. FIG. 15 shows the modification interference value of phosphate alkylation. Data are normalized to A17 3 'phosphate. FIG. 16 shows the modification interference values for DMS modification of N3C and NlA. Data are normalized to C36; A34. FIG. 17 shows modification interference values for ketoxal modification of N1G and N2G. Data is normalized to G5. FIG. 18 shows modification interference values for CMCT modification of N3U and N1G. Data is standardized for U38. FIG. 19 shows the modification interference values for DEPC modification of N7A and N7G. Data are normalized to G19; A34. FIG. 20 shows the chemical modification of RNA ligands in the presence of HIV-1 Rev protein. Positions are shown that show either reduced modification or enhanced modification in the presence of protein as compared to modification under non-denaturing conditions but without protein. FIG. 21 shows adjacent 5 'and 3' arrays of "6a" biased random regions used in SELEX. The template that produces the initial RNA population is the oligonucleotide:

（ここで、鋳型オリゴの小文字は、各位置で６２．５％の小文字のものと各１２．５％の他の３つのヌクレオチドの量での試薬の混合物を合成に使用したことを示す）から作成された。６ａ配列の下に記載したのは、このＲＮＡの集団を有するＲｅｖタンパクを用いてセレックスを６回実施した後にクローン化した３８単離体の配列である。これらの単離体に見い出される６ａ配列との差は太字で示してある。下線部は、モチーフＩ Ｒｅｖリガンドの隆起に隣接する軸を包含する予測された塩基対である。５’と３’の固定隣接配列から含まれる塩基は小文字である。
図２１は、セレックスで使用された「６ａ」の偏ったランダム領域の隣接する５’と３’配列を示す。最初のＲＮＡ集団を産生する鋳型は、下記のオリゴヌクレオチド：

(Here, the lower case letters of the template oligo indicate that a 62.5% lower case letter at each position and a mixture of reagents in the amount of the other 3 nucleotides each 12.5% were used in the synthesis) Created. Listed below the 6a sequence is the sequence of 38 isolates cloned after six SELEX runs using Rev protein with this population of RNA. Differences from the 6a sequence found in these isolates are shown in bold. The underlined portion is the predicted base pair encompassing the axis adjacent to the motif I Rev ligand ridge. Bases included from 5 ′ and 3 ′ fixed flanking sequences are lower case.
FIG. 21 shows adjacent 5 ′ and 3 ′ sequences of the “6a” biased random region used in SELEX. The template that produces the initial RNA population is the oligonucleotide:

（ここで、鋳型オリゴの小文字は、各位置で６２．５％の小文字のものと各１２．５％の他の３つのヌクレオチドの量での試薬の混合物を合成に使用したことを示す）から作成された。６ａ配列の下に記載したのは、このＲＮＡの集団を有するＲｅｖタンパクを用いてセレックスを６回実施した後にクローン化した３８単離体の配列である。これらの単離体に見い出される６ａ配列との差は太字で示してある。下線部は、モチーフＩ Ｒｅｖリガンドの隆起に隣接する軸を包含する予測された塩基対である。５’と３’の固定隣接配列から含まれる塩基は小文字である。
図２２は：Ａ）図２１に見い出された配列の集合中の、Ｒｅｖ ６ａリガンド配列の対応する位置に見い出された各ヌクレオチドの数；Ｂ）これらの位置に見い出された各ヌクレオチドの小数で示した頻度（ｘ÷３８、ここでｘは１からの数）；そしてＣ）Ｂ）の小数で示した頻度と、鋳型合成の間の投入したオリゴヌクレオチドの混合物に基づく予測頻度との差［「野生型」の位置については、（ｘ÷３８）−０．６２５、そして代替配列については、（ｘ÷３８）−０．１２５］、を含む３セットの表を示す。 図２２は：Ａ）図２１に見い出された配列の集合中の、Ｒｅｖ ６ａリガンド配列の対応する位置に見い出された各ヌクレオチドの数；Ｂ）これらの位置に見い出された各ヌクレオチドの小数で示した頻度（ｘ÷３８、ここでｘは１からの数）；そしてＣ）Ｂ）の小数で示した頻度と、鋳型合成の間の投入したオリゴヌクレオチドの混合物に基づく予測頻度との差［「野生型」の位置については、（ｘ÷３８）−０．６２５、そして代替配列については、（ｘ÷３８）−０．１２５］、を含む３セットの表を示す。 図２３は：Ａ）図２１に見い出された配列の集合中の、Ｒｅｖ ６ａリガンド配列の対応する位置に見い出された各ヌクレオチドの数；Ｂ）これらの位置に見い出された各ヌクレオチドの小数で示した頻度（ｘ÷３８、ここでｘはＡからの数）；そしてＣ）Ｂ）の小数で示した頻度と、鋳型合成の間の投入したオリゴヌクレオチドの混合物に基づく予測頻度との差［「野生型」の位置については、（ｘ÷３８）−０．３９５；１つの代替ヌクレオチドと１つの野生型ヌクレオチドを含有する塩基対については、（ｘ÷３８）−０．０７８；そして１つの代替ヌクレオチドの塩基対については（ｘ÷３８）−０．０１６］、を含む３セットの表を示す。値はプリンとピリミジン対のみが示され、他の８つのピリミジンとプリン対は集合的に数えて「その他」として示し、セクションＣ）に（ｘ÷３８）−０．２５２として計算してある。 図２３は：Ａ）図２１に見い出された配列の集合中の、Ｒｅｖ ６ａリガンド配列の対応する位置に見い出された各ヌクレオチドの数；Ｂ）これらの位置に見い出された各ヌクレオチドの小数で示した頻度（ｘ÷３８、ここでｘはＡからの数）；そしてＣ）Ｂ）の小数で示した頻度と、鋳型合成の間の投入したオリゴヌクレオチドの混合物に基づく予測頻度との差［「野生型」の位置については、（ｘ÷３８）−０．３９５；１つの代替ヌクレオチドと１つの野生型ヌクレオチドを含有する塩基対については、（ｘ÷３８）−０．０７８；そして１つの代替ヌクレオチドの塩基対については（ｘ÷３８）−０．０１６］、を含む３セットの表を示す。値はプリンとピリミジン対のみが示され、他の８つのピリミジンとプリン対は集合的に数えて「その他」として示し、セクションＣ）に（ｘ÷３８）−０．２５２として計算してある。 図２４は、あらかじめ決定されたＲｅｖタンパクリガンドモチーフ１コンセンサス（１９９１年６月１０日に出願した、合衆国特許出願番号０７／７１４，１３１号；ＷＯ９１／１９８１３号）を示す。同じ出願から６ａ配列、および好ましいコンセンサスは、図２２と２３に与えられるデータから解釈されるように、偏ったランダム化セレックスにより誘導される。好ましいコンセンサス中の絶対的に保存される位置は太字で示し、そしてＳ−Ｓ’はＣ−ＧまたはＧ−Ｃ塩基対を示す。 図２５は、ｔａｔタンパクと相互作用するＨＩＶ−１からの天然ＲＮＡ配列（すなわちＴＡＲＲＮＡ）を示す。配列の四角で囲んだ領域は、ｔａｔ−ＴＡＲ相互作用において重要であることが見い出されたヌクレオチドである。 図２６は、ＨＩＶ−１ ｔａｔタンパクに対する核酸リガンドとして本発明により単離されたリガンドの配列を示す。これらの配列は、３つのモチーフ（Ｉ、ＩＩおよびＩＩＩ）中の共通の２次構造と１次配列により分類される。ＲＮＡらせんを予測させる逆繰り返し配列を、矢印で示す。各モチーフ中の一次配列の相同の領域は、破線の四角で囲まれている。高い親和性結合に必須の配列情報の境界は、実線の四角で示してある。配列１と１７は、３っの同定されたモチーフのいずれにも適合しない。 図２７は、図２６に示す各リガンドモチーフのコンセンサス２次構造と一次配列の概略図を示す。Ｘは非保存ヌクレオチドの位置を示す。Ｘ’はらせん中のその位置でのＸに相補的な塩基対を示し、Ｒはプリンを、そしてＹはピリミジンを示す。モチーフＩＩＩ中の破線は、そのループの部分での可変数のヌクレオチドを示す。 図２８は、図２６から選択されたリガンドＲＮＡ、および４０ｎ ＲＮＡ候補混合物のニトロセルロースフィルターへの、ｔａｔタンパク濃度依存性の結合を示す。 図２９は、セレックスにより単離されたヒト・トロンビン・タンパク（シグマ（Ｓｉｇｍａ））に対するＲＮＡリガンドのヌクレオチド配列（配列ＩＤ番号：１３７−１５５）を示す。各配列は、左から右へ３つのブロック：１）５’固定領域、２）３０Ｎ可変領域、および３）３’固定領域に分画される。個々の配列は、下線付の太字で示すように、３０Ｎ可変領域内の保存配列モチーフにより、クラスＩとクラスＩＩに分類される。 図３０は、ＲＮＡリガンド（配列ＩＤ番号：１５６−１５９）の提案される２次構造を示す。（Ａ）は、７６ヌクレオチドのクラスＩ ＲＮＡクローン６、１６、および１８の配列、そしてクラスＩＩの７２ヌクレオチドのクローン２７の配列を示す。境界位置（［は５’境界を意味し、そして］は３’境界を意味する）も、示してある。境界実験から決定された、各ＲＮＡの可能な２次構造が、（Ｂ）に示してある。境界は下線で示される。（Ａ）と（Ｂ）には５’と３’の固定領域を小文字で示し、３０Ｎランダム領域は大文字で、そして保存領域は太字の大文字で示してある。合成されたヘアピン構造は、記載したヌクレオチドの総数と一緒に四角で囲んである。 図３０は、ＲＮＡリガンド（配列ＩＤ番号：１５６−１５９）の提案される２次構造を示す。（Ａ）は、７６ヌクレオチドのクラスＩ ＲＮＡクローン６、１６、および１８の配列、そしてクラスＩＩの７２ヌクレオチドのクローン２７の配列を示す。境界位置（［は５’境界を意味し、そして］は３’境界を意味する）も、示してある。境界実験から決定された、各ＲＮＡの可能な２次構造が、（Ｂ）に示してある。境界は下線で示される。（Ａ）と（Ｂ）には５’と３’の固定領域を小文字で示し、３０Ｎランダム領域は大文字で、そして保存領域は太字の大文字で示してある。合成されたヘアピン構造は、記載したヌクレオチドの総数と一緒に四角で囲んである。 図３１は、トロンビンのリガンドの結合曲線を示す。ヒト・トロンビン（シグマ（Ｓｉｇｍａ））との結合分析のために、（Ａ）で、クラスＩから３つ：ＲＮＡ６、ＲＮＡ１６、およびＲＮＡ１８、およびクラスＩＩから１つ：ＲＮＡ２７を含む、ユニークな３０Ｎ配列モチーフを有するＲＮＡを選択した。３０Ｎ３候補混合物の大量のＲＮＡ配列の結合も、示してある。（Ｂ）では、クラスＩ ＲＮＡクローン６、１６、１８、およびクラスＩＩ ＲＮＡクローン２７の結合を示すが、エンザイムリサーチラボラトリーズ（Ｅｎｚｙｍｅ Ｒｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）からのヒト・トロンビンとの結合を示す。（Ｃ）では、（Ｂ）と同一の条件下での、１５塩基のｓｓＤＮＡ５’−ＧＧＴＴＧＧＴＧＴＧＧＴＴＧＧ−３’（Ｇ１５Ｄ）（配列ＩＤ番号：１）、クラスＩクローン１６ヘアピン構造（２４Ｒ、３９Ｄ）およびクラスＩＩクローン２７ヘアピン構造（３３Ｒ）の結合を示す（図３０Ｂ参照）。ＲＮＡヘアピン構造の場合は、ＲはＲＮＡ合成を意味し、そしてＤはＤＮＡ鋳型からの転写を意味する。 図３２は、未修飾ＲＮＡと、２’−ＮＨ_２リボースヌクレオチドを含有するように修飾されたピリミジンを有するＲＮＡとの間の、ＲＮＡリガンドの結合比較を示す。（Ａ）大量ＲＮＡ３０Ｎ候補混合物と２−ＮＨ_２修飾３０Ｎ候補混合物、（Ｂ）クラスＩ ＲＮＡ１６と２−ＮＨ_２修飾ＲＮＡ１６、および（Ｃ）クラスＩＩ ＲＮＡ２７と２−ＮＨ２修飾ＲＮＡ２７の結合比較が示してある。 図３３は、本発明の１５塩基のｓｓＤＮＡ Ｇ１５ＤとＲＮＡヘアピンリガンド間の、ヒト・トロンビンへの結合に関する競合実験を示す。Ａ）では、トレーサーＧ１５Ｄの濃度はタンパク濃度１μＭに等しい。結合の競合物は、Ｇ１５Ｄ自体、クラスＩ ＲＮＡ１６からの２４と３９ヌクレオチドのＲＮＡヘアピン構造、およびクラスＩＩ ＲＮＡ２７からの３３ヌクレオチドＲＮＡヘアピン構造を含む（図３０参照）。結合は、結合したＧ１５Ｄの相対割合として表してあり、これは競合物のある時のＧ１５Ｄ結合と、競合物のない時のＧ１５Ｄ結合との比である。Ｂ）では、ＲＮＡ３３はトレーサーであり、このトレーサーの濃度はプロテアーゼの濃度３００ｎＭに等しい。結合の競合物は、ｓｓＤＮＡ Ｇ１５ＤとＲＮＡ２４を含む。 図３４は、記載されたＲＮＡリガンド阻害剤の存在下および非存在下での、トロンビンの機能の測定の結果を示す。Ａ）では、記載したトロンビンとＲＮＡ濃度での、発色性基質Ｓ−２２３８（Ｈ−Ｄ−Ｐｈｅ−Ｐｉｐ−Ａｒｇ−ｐニトロアニリン）のトロンビンによる加水分解を、ＯＤ４０５での変化により測定した。Ｂ）では、フィブリノーゲンのフィブリンへの変換と、その結果生じる凝固形成を、記載されたＲＮＡリガンド阻害剤の存在下および非存在下での傾斜試験１５（ｔｉｌｔ ｔｅｓｔ）により測定した。 図３５は、トロンビンのリガンドに対する結合の特異性を示す。Ａ）ヒト・アンチトロンビンＩＩＩ（シグマ（Ｓｉｇｍａ））、およびＢ）ヒト・プロトロンビン（シグマ）との結合分析のために、クラスＩ ＲＮＡ１６、クラスＩＩ ＲＮＡ２７、および大量の３０Ｎ３ ＲＮＡを選択した。 図３６は、ファミリー１リガンド７Ａ（△）、ファミリー２リガンド１２Ａ（□）、セレックス実験ＡのランダムＲＮＡ（＋）およびセレックス実験ＢのランダムＲＮＡ（×）の結合曲線を示す。ニトロセルロースフィルターに結合したＲＮＡの画分を、遊離のタンパク濃度の関数としてプロットして、データの点を式２に適合させた。以下の濃度のＲＮＡを使用した：７Ａと１２Ａでは＜１００ｐＭ、ランダムＲＮＡでは１０ｎＭ。結合反応は、０．０１％ヒト血清アルブミンを含有するリン酸緩衝化生理食塩水中で３７℃で行った。 図３７は、低親和性（パネルＡ）と高親和性（パネルＢ）細胞表面リセプターへの^１２５ Ｉ−ｂＦＧＦの結合に及ぼす、リガンド５Ａ（○）、７Ａ（△）、１２Ａ（□）、２６Ａ（菱形）、セレックス実験ＡのランダムＲＮＡ（＋）およびセレックス実験ＢのランダムＲＮＡ（×）の効果を示す。実験は、基本的にログハニとモスカテリ（Ｒｏｇｈａｎｉ ＆ Ｍｏｓｃａｔｅｌｌｉ）（１９９２） Ｊ．Ｂｉｏｌ．Ｃｈｅｍ． ２６７：２２１５６に記：載されたように行った。 図３７は、低親和性（パネルＡ）と高親和性（パネルＢ）細胞表面リセプターへの^１２５ Ｉ−ｂＦＧＦの結合に及ぼす、リガンド５Ａ（○）、７Ａ（△）、１２Ａ（□）、２６Ａ（菱形）、セレックス実験ＡのランダムＲＮＡ（＋）およびセレックス実験ＢのランダムＲＮＡ（×）の効果を示す。実験は、基本的にログハニとモスカテリ（Ｒｏｇｈａｎｉ ＆ Ｍｏｓｃａｔｅｌｌｉ）（１９９２） Ｊ．Ｂｉｏｌ．Ｃｈｅｍ． ２６７：２２１５６に記：載されたように行った。 図３８は、^３２Ｐ−標識ＲＮＡリガンド５Ａ（○）、７Ａ（△）、１２Ａ（□）および２６Ａ（菱形）の、ヘパリン（平均分子量５，０００Ｄａ）による競合置換を示す。ニトロセルロースフィルターに結合した全体の投入ＲＮＡのパーセントを、ヘパリン濃度の関数としてプロットする。実験は、０．０１％ヒト血清アルブミン、０．３μＭのＲＮＡ、および３０ｎＭのｂＦＧＦを含有するリン酸緩衝化生理食塩水中で３７℃で行った。 図３９は、高い親和性でｂＦＧＦに結合するファミリー１リガンドの提唱された２次構造を示す。矢印は、保存ループに隣接する２本鎖（軸）領域を示す。小文字は、定常領域にあるヌクレオチドを示す。 図４０は、ファミリー１リガンドの提唱された二次構造を示す。 図４１は、ファミリー１とファミリー２リガンドのコンセンサス構造を示す。Ｙ＝ＣまたはＵ；Ｒ＝ＡまたはＧ；Ｗ＝ＡまたはＵ；Ｈ＝Ａ、Ｕ、またはＣ；Ｄ＝Ａ、Ｇ、またはＵ；Ｎ＝任意の塩基である。相補塩基がプライムされる。カッコ内の記号は、その位置で下付文字で示した境界範囲内の可変数の塩基または塩基対を示す。

(Here, the lower case letters of the template oligo indicate that a 62.5% lower case letter at each position and a mixture of reagents in the amount of the other 3 nucleotides each 12.5% were used in the synthesis) Created. Listed below the 6a sequence is the sequence of 38 isolates cloned after six SELEX runs using Rev protein with this population of RNA. Differences from the 6a sequence found in these isolates are shown in bold. The underlined portion is the predicted base pair encompassing the axis adjacent to the motif I Rev ligand ridge. Bases included from 5 ′ and 3 ′ fixed flanking sequences are lower case.
Figure 22: A) Number of each nucleotide found in the corresponding position of the Rev 6a ligand sequence in the set of sequences found in Figure 21; B) Decimal number of each nucleotide found in these positions Difference between the frequency expressed as a fraction of the frequency (x ÷ 38, where x is a number from 1); and C) B) and the predicted frequency based on the mixture of oligonucleotides input during template synthesis [“ Three sets of tables are shown, including (x ÷ 38) −0.625 for the “wild type” position and (x ÷ 38) −0.125] for the alternative sequence. Figure 22: A) Number of each nucleotide found in the corresponding position of the Rev 6a ligand sequence in the set of sequences found in Figure 21; B) Decimal number of each nucleotide found in these positions Difference between the frequency expressed as a fraction of the frequency (x ÷ 38, where x is a number from 1); and C) B) and the predicted frequency based on the mixture of oligonucleotides input during template synthesis [“ Three sets of tables are shown, including (x ÷ 38) −0.625 for the “wild type” position and (x ÷ 38) −0.125] for the alternative sequence. FIG. 23 shows: A) the number of each nucleotide found at the corresponding position of the Rev 6a ligand sequence in the set of sequences found in FIG. 21; B) the fraction of each nucleotide found at these positions. Difference between the frequency expressed as a fraction of the frequency (x ÷ 38, where x is a number from A); and C) B) and the predicted frequency based on the mixture of input oligonucleotides during template synthesis [“ (X ÷ 38) −0.395 for the “wild type” position; (x ÷ 38) −0.078 for the base pair containing one alternative nucleotide and one wild type nucleotide; and one alternative For nucleotide base pairs, (x ÷ 38) −0.016], three sets of tables are shown. Values are shown only for purine and pyrimidine pairs, and the other eight pyrimidine and purine pairs are collectively counted as “others” and are calculated in section C) as (x ÷ 38) −0.252. FIG. 23 shows: A) the number of each nucleotide found at the corresponding position of the Rev 6a ligand sequence in the set of sequences found in FIG. 21; B) the fraction of each nucleotide found at these positions. Difference between the frequency expressed as a fraction of the frequency (x ÷ 38, where x is a number from A); and C) B) and the predicted frequency based on the mixture of input oligonucleotides during template synthesis [“ (X ÷ 38) −0.395 for the “wild type” position; (x ÷ 38) −0.078 for the base pair containing one alternative nucleotide and one wild type nucleotide; and one alternative For nucleotide base pairs, (x ÷ 38) −0.016], three sets of tables are shown. Values are shown only for purine and pyrimidine pairs, and the other eight pyrimidine and purine pairs are collectively counted as “others” and are calculated in section C) as (x ÷ 38) −0.252. FIG. 24 shows the predetermined Rev protein ligand motif 1 consensus (US Patent Application No. 07 / 714,131; WO 91/19813, filed June 10, 1991). The 6a sequence from the same application, and the preferred consensus, is derived by biased randomized SELEX as interpreted from the data given in FIGS. Absolutely conserved positions in the preferred consensus are shown in bold, and SS ′ indicates CG or GC base pairs. FIG. 25 shows the native RNA sequence from HIV-1 that interacts with the tat protein (ie TARRNA). The boxed region of the sequence is the nucleotide that was found to be important in the tat-TAR interaction. FIG. 26 shows the sequence of a ligand isolated according to the present invention as a nucleic acid ligand for HIV-1 tat protein. These sequences are classified by the common secondary structure and primary sequence in the three motifs (I, II and III). An inverted repeat that predicts an RNA helix is indicated by an arrow. The region of homology of the primary sequence in each motif is surrounded by a dashed box. The boundaries of the sequence information essential for high affinity binding are indicated by solid squares. Sequences 1 and 17 do not match any of the three identified motifs. FIG. 27 shows a schematic diagram of the consensus secondary structure and primary sequence of each ligand motif shown in FIG. X indicates the position of the non-conserved nucleotide. X ′ represents a base pair complementary to X at that position in the helix, R represents a purine, and Y represents a pyrimidine. The dashed line in motif III indicates a variable number of nucleotides in the portion of the loop. FIG. 28 shows tat protein concentration-dependent binding of ligand RNA selected from FIG. 26 and a 40 n RNA candidate mixture to a nitrocellulose filter. FIG. 29 shows the nucleotide sequence (SEQ ID NO: 137-155) of the RNA ligand for human thrombin protein (Sigma) isolated by SELEX. Each sequence is partitioned into three blocks from left to right: 1) 5 ′ fixed region, 2) 30N variable region, and 3) 3 ′ fixed region. Individual sequences are classified into class I and class II by conserved sequence motifs in the 30N variable region, as shown in bold underlined. FIG. 30 shows the proposed secondary structure of the RNA ligand (SEQ ID NO: 156-159). (A) shows the sequence of 76 nucleotide class I RNA clones 6, 16, and 18 and the sequence of class II 72 nucleotide clone 27. Boundary positions ([means 5 'boundary and] means 3' boundary) are also shown. Possible secondary structures of each RNA determined from boundary experiments are shown in (B). The border is underlined. In (A) and (B), the 5 ′ and 3 ′ fixed areas are shown in lower case, the 30N random area is shown in upper case, and the storage area is shown in bold upper case. The synthesized hairpin structure is boxed with the total number of nucleotides listed. FIG. 30 shows the proposed secondary structure of the RNA ligand (SEQ ID NO: 156-159). (A) shows the sequence of 76 nucleotide class I RNA clones 6, 16, and 18 and the sequence of class II 72 nucleotide clone 27. Boundary positions ([means 5 'boundary and] means 3' boundary) are also shown. Possible secondary structures of each RNA determined from boundary experiments are shown in (B). The border is underlined. In (A) and (B), the 5 ′ and 3 ′ fixed areas are shown in lower case, the 30N random area is shown in upper case, and the storage area is shown in bold upper case. The synthesized hairpin structure is boxed with the total number of nucleotides listed. FIG. 31 shows the binding curve of thrombin ligand. For binding analysis with human thrombin (Sigma), in (A), a unique 30N sequence comprising three from class I: RNA6, RNA16, and RNA18, and one from class II: RNA27. RNA with a motif was selected. The binding of large amounts of RNA sequences from the 30N3 candidate mixture is also shown. (B) shows binding of class I RNA clones 6, 16, 18 and class II RNA clone 27, but binding to human thrombin from Enzyme Research Laboratories. (C) 15 base ssDNA 5′-GGTTGGGTTGGTTGG-3 ′ (G15D) (SEQ ID NO: 1), class I clone 16 hairpin structure (24R, 39D) and class under the same conditions as in (B) Illustrates binding of II clone 27 hairpin structure (33R) (see FIG. 30B). In the case of RNA hairpin structures, R means RNA synthesis and D means transcription from a DNA template. FIG. 32 shows an RNA ligand binding comparison between unmodified RNA and RNA with pyrimidines modified to contain 2′-NH ₂ ribose nucleotides. (A) Large RNA 30N candidate mixture and 2-NH ₂ modified 30N candidate mixture, (B) Class I RNA 16 and 2-NH ₂ modified RNA 16 and (C) Class II RNA 27 and 2-NH ₂ modified RNA 27 binding comparisons are shown. is there. FIG. 33 shows a competition experiment for binding to human thrombin between the 15 base ssDNA G15D of the present invention and an RNA hairpin ligand. In A), the concentration of tracer G15D is equal to a protein concentration of 1 μM. Binding competitors include G15D itself, 24 and 39 nucleotide RNA hairpin structures from class I RNA16, and 33 nucleotide RNA hairpin structures from class II RNA27 (see FIG. 30). Binding is expressed as the relative percentage of G15D bound, which is the ratio of G15D binding in the presence of competitor to G15D binding in the absence of competitor. In B) RNA 33 is a tracer, the concentration of which is equal to the protease concentration of 300 nM. Binding competitors include ssDNA G15D and RNA24. FIG. 34 shows the results of measurement of thrombin function in the presence and absence of the described RNA ligand inhibitors. In A), the thrombin hydrolysis of the chromogenic substrate S-2238 (HD-Phe-Pip-Arg-pnitroaniline) at the indicated thrombin and RNA concentrations was measured by a change in OD405. In B), the conversion of fibrinogen to fibrin and the resulting coagulation formation was measured by the tilt test 15 in the presence and absence of the described RNA ligand inhibitors. FIG. 35 shows the specificity of binding of thrombin to the ligand. Class I RNA 16, Class II RNA 27, and large quantities of 30N3 RNA were selected for binding analysis with A) human antithrombin III (Sigma), and B) human prothrombin (Sigma). FIG. 36 shows binding curves for Family 1 ligand 7A (Δ), Family 2 ligand 12A (□), SELEX experiment A random RNA (+) and SELEX experiment B random RNA (×). The fraction of RNA bound to the nitrocellulose filter was plotted as a function of free protein concentration and the data points were fitted to Equation 2. The following concentrations of RNA were used: <100 pM for 7A and 12A, 10 nM for random RNA. The binding reaction was performed at 37 ° C. in phosphate buffered saline containing 0.01% human serum albumin. FIG. 37 shows the effects of ligand 5A (◯), 7A (Δ), 12A (□), 26A on the binding of ¹²⁵ I-bFGF to low affinity (panel A) and high affinity (panel B) cell surface receptors. (Diamonds), effects of random RNA (+) from SELEX experiment A and random RNA (X) from SELEX experiment B are shown. The experiment is basically based on Loghani & Moscatelli (1992) J. MoI. Biol. Chem. 267: 22156: as described. FIG. 37 shows the effects of ligand 5A (◯), 7A (Δ), 12A (□), 26A on the binding of ¹²⁵ I-bFGF to low affinity (panel A) and high affinity (panel B) cell surface receptors. (Diamonds), effects of random RNA (+) from SELEX experiment A and random RNA (X) from SELEX experiment B are shown. The experiment is basically based on Loghani & Moscatelli (1992) J. MoI. Biol. Chem. 267: 22156: as described. FIG. 38 shows competitive displacement of ³² P-labeled RNA ligands 5A (◯), 7A (Δ), 12A (□), and 26A (diamond) by heparin (average molecular weight 5,000 Da). The percentage of total input RNA bound to the nitrocellulose filter is plotted as a function of heparin concentration. Experiments were performed at 37 ° C. in phosphate buffered saline containing 0.01% human serum albumin, 0.3 μM RNA, and 30 nM bFGF. FIG. 39 shows the proposed secondary structure of a family 1 ligand that binds bFGF with high affinity. Arrows indicate double stranded (axial) regions adjacent to the conserved loop. Lower case letters indicate nucleotides in the constant region. FIG. 40 shows the proposed secondary structure of Family 1 ligands. FIG. 41 shows the consensus structure of family 1 and family 2 ligands. Y = C or U; R = A or G; W = A or U; H = A, U, or C; D = A, G, or U; N = any base. Complementary bases are primed. Symbols in parentheses indicate a variable number of bases or base pairs within the boundary range indicated by the subscript at that position.

Claims (83)

A method for producing an improved nucleic acid ligand, which is a target ligand, from a candidate mixture of nucleic acids,
a) contacting the candidate mixture with a target, wherein nucleic acids having increased affinity for the target relative to the candidate mixture may be fractionated from the remainder of the candidate mixture;
b) fractionating the nucleic acid with increased affinity from the remainder of the candidate mixture;
c) amplifying nucleic acid with increased affinity to obtain a ligand enriched mixture of nucleic acids;
d) repeat steps a) -c) if necessary to identify nucleic acid ligands;
e) measuring a nucleic acid residue of a nucleic acid ligand that is essential for binding to the target; and f) producing the improved nucleic acid ligand based on the measurement. Measurement is
2. preparing a modified nucleic acid that is identical to the nucleic acid ligand except for a single residue substitution; and b) assessing the binding affinity of the modified nucleic acid ligand relative to the nucleic acid ligand. The method described in 1. Measurement is
a) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for the absence of one or more terminal residues; and b) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand. The method of claim 1. Measurement is
2. The method of claim 1, comprising: a) preparing a modified nucleic acid by chemically modifying the nucleic acid ligand; and b) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand. Measurement is
2. The method of claim 1, comprising a) chemically modifying the nucleic acid ligand in the presence of the target; and b) determining which nucleic acid residues are not modified. A method for producing an improved nucleic acid ligand, which is a target ligand, from a candidate mixture of nucleic acids,
a) contacting the candidate mixture with the target, wherein nucleic acids having increased affinity for the target relative to the candidate mixture may be fractionated from the remainder of the candidate mixture;
b) fractionating the nucleic acid with increased affinity from the remainder of the candidate mixture;
c) amplifying nucleic acid with increased affinity to obtain a ligand enriched mixture of nucleic acids;
d) repeat steps a) -c) if necessary to identify the nucleic acid ligand;
e) measuring the three-dimensional structure of the nucleic acid ligand; and f) producing the improved nucleic acid ligand based on the measurement. Measurement is
a) denature the nucleic acid ligand;
and b) chemically modifying the denatured and non-denatured nucleic acid ligands; and c) determining which nucleic acid residues that are not modified in the non-denatured nucleic acid ligand are modified in the denatured nucleic acid ligand. 6. The method according to 6. Measurement is
7. The method of claim 6, comprising a) performing a covariance analysis on the nucleic acid ligand. A method for producing an improved nucleic acid ligand for a target from a plurality of nucleic acid ligands, comprising:
measuring the nucleic acid residue of the nucleic acid ligand that is essential for binding to the target; and b) measuring the three-dimensional structure of the nucleic acid ligand. A method for identifying an elongated nucleic acid ligand that is a target ligand from a candidate mixture of nucleic acids, comprising:
a) contacting the candidate mixture with a target, wherein nucleic acids having increased affinity for the target relative to the candidate mixture are fractionated from the remainder of the candidate mixture;
b) the nucleic acid with increased affinity may be fractionated from the remainder of the candidate mixture;
c) amplifying nucleic acid with increased affinity to obtain a ligand enriched mixture of nucleic acids;
d) repeat steps a) -c) if necessary to identify the nucleic acid ligand;
e) creating a secondary candidate mixture consisting of nucleic acids having a fixed region and a randomized region, wherein the fixed region corresponds to the nucleic acid ligand identified in d) above;
f) contacting the secondary candidate mixture with the target, wherein nucleic acids having increased affinity for the target relative to the candidate mixture may be fractionated from the remainder of the candidate mixture;
g) amplifying the nucleic acid with increased affinity to obtain a ligand enriched mixture of nucleic acids; and h) repeating steps e) -g) if necessary to identify the nucleic acid ligand, Method.   A non-naturally occurring nucleic acid ligand for HIV-RT protein.   The nucleic acid ligand according to claim 11, which is produced by the method according to claim 1. A nucleic acid ligand according to claim 12, comprising:
d) repeat steps a) -c) if necessary to identify the nucleic acid ligand;
e) measuring the nucleic acid residue of a nucleic acid ligand that is essential for binding to the HIV-RT; and f) producing the improved nucleic acid ligand based on the measurement, the ligand comprising an additional step.   The nucleic acid ligand according to claim 11, which is produced by the method according to claim 2.   The nucleic acid ligand according to claim 11, which is produced by the method according to claim 3.   The nucleic acid ligand according to claim 11, which is produced by the method according to claim 4.   The nucleic acid ligazod according to claim 11, which is produced by the method according to claim 5. Array:

The nucleic acid ligand according to claim 11, wherein XX ′ represents a preferred base pair in the sequence. Array:

12. The nucleic acid ligand of claim 11, having a sequence that is substantially homologous to (wherein XX 'represents a preferred base pair) and has substantially the same ability to bind to HIV-RT. .   12. The nucleic acid of claim 11 identified by the method of claim 10. Array:

21. A nucleic acid ligand according to claim 20, wherein Z is selected from the group consisting of the sequences shown in FIG. 9 (SEQ ID NO: 115-135) [10]. Array:

(In the sequence, Z is substantially homologous to FIG. 9 (selected from the group consisting of the sequences shown in SEQ ID NO: 115-135) and has substantially the same ability to bind to HIV-RT. 21. The nucleic acid ligand of claim 20, having a sequence having   The nucleic acid ligand according to claim 21, wherein Z is selected from the group consisting of extension motif I and extension motif II, each shown in Figure 9 (SEQ ID NO: 115-135).   23. The nucleic acid ligand according to claim 22, wherein Z is selected from the group consisting of extension motif I and extension motif II, each shown in FIG. 9 (SEQ ID NO: 115-135).   A non-naturally occurring nucleic acid ligand of the HIV-1 Rev protein.   26. The nucleic acid ligand of claim 25 produced by the method of claim 1.   26. The nucleic acid ligand of claim 25 produced by the method of claim 2.   The nucleic acid ligand according to claim 25, which is produced by the method according to claim 3.   26. The nucleic acid ligand of claim 25, produced by the method of claim 4.   26. The nucleic acid ligand of claim 25, produced by the method of claim 5. Array:

26. The nucleic acid ligand of claim 25 having Array:

26. The nucleic acid ligand of claim 25 having a sequence that is substantially homologous to and having substantially the same ability to bind to HIV-1 Rev protein. A method for producing an improved ligand for a target comprising:
(A) preparing a candidate mixture of nucleic acids;
(B) contacting the candidate mixture with a target, wherein nucleic acids having increased affinity for the target relative to the candidate mixture may be fractionated from the remainder of the candidate mixture;
(C) fractionating the nucleic acid with increased affinity from the remainder of the candidate mixture;
(D) amplifying nucleic acids with increased affinity to obtain a ligand-enriched mixture of nucleic acids;
(E) repeating steps (b)-(d) if necessary to identify the nucleic acid ligand;
(F) measuring a nucleic acid residue of a nucleic acid ligand that is essential for binding to the target;
(G) measuring the three-dimensional structure of the nucleic acid ligand; and (h) designing the improved ligand based on the measurement. A method of identifying a nucleic acid ligands to HIV-1 tat protein,
a) preparing a candidate mixture of nucleic acids;
b) fractionating members of the candidate mixture based on affinity to the protein; and c) amplifying molecules selected from candidate mixtures having a relatively high affinity for the protein and comparing to the protein Obtaining a mixture of nucleic acids enriched in sequences having high affinity.   35. The method of claim 34, further comprising d) repeating steps b) and c).   35. The method of claim 34, wherein the candidate mixture of nucleic acids consists of single stranded nucleic acids.   37. The method of claim 36, wherein the candidate mixture consists of RNA. 35. A nucleic acid ligand for HIV-1 tat protein identified by the method of claim 34.   39. The nucleic acid ligand of claim 38, which is a single stranded nucleic acid.   40. The nucleic acid ligand of claim 38, which is a single stranded RNA sequence. A purified, isolated, non-naturally occurring nucleic acid ligand for the HIV-1 tat protein.   42. The nucleic acid ligand of claim 41, wherein the ligand is selected from the group consisting of the sequence shown in FIG. 42. The nucleic acid ligand of claim 41, wherein the ligand is substantially homologous to a ligand selected from the group consisting of the sequence shown in FIG. 26 and has substantially the same ability to bind to the tat protein.   42. The nucleic acid ligand of claim 41, wherein the ligand is selected from the group consisting of motif I, motif II and motif III shown in FIG. 28. The ligand is substantially homologous to a ligand selected from the group consisting of motif I, motif II and motif III shown in FIG. 27 and has substantially the same ability to bind to the tat protein. 42. The nucleic acid ligand according to 41.   42. The nucleic acid ligand of claim 41, wherein the ligand is chemically modified at the ribose and / or phosphate and / or base positions. 42. The nucleic acid ligand of claim 41, wherein the ligand has substantially the same structure and substantially the same ability to bind to a tat protein as a ligand selected from the group consisting of the sequence shown in FIG. .   The ligand has substantially the same structure and substantially the same ability to bind to the tat protein as a ligand selected from the group consisting of motif I, motif II and motif III shown in FIG. 42. The nucleic acid ligand of claim 41. A method of identifying a nucleic acid rigaside for thrombin, comprising:
a) preparing a candidate mixture of RNA nucleic acids;
b) contacting the candidate mixture with thrombin, wherein nucleic acids with increased affinity for thrombin may be fractionated from the remainder of the candidate mixture;
c) fractionating members of the candidate mixture based on affinity to thrombin; and d) amplifying a molecule selected from the candidate mixture having a relatively high affinity for thrombin to Obtaining a mixture of nucleic acids enriched in sequences having a high affinity.   50. The method of claim 49, further comprising e) repeating steps b), c) and d).   50. The method of claim 49, wherein the candidate mixture of RNA nucleic acids comprises single stranded nucleic acids.   50. An RNA nucleic acid ligand for thrombin identified by the method of claim 49.   53. The nucleic acid ligand of claim 52, which is a single stranded nucleic acid.   A purified, isolated, non-naturally occurring RNA ligand for thrombin.   55. The RNA ligand of claim 54, wherein the ligand is selected from the group consisting of the sequence shown in Figure 29 (SEQ ID NO: 137-154).   30. The ligand is substantially homologous to a ligand selected from the group consisting of the sequences shown in FIG. 29 (SEQ ID NO: 137-154) and has substantially the same ability to bind thrombin. 54. The RNA ligand according to 54.   55. The RNA ligand of claim 54, wherein the ligand is chemically modified at the ribose and / or phosphate and / or base positions.   55. The RNA of claim 54, wherein the ligand has substantially the same structure as the sequence shown in Figure 29 (SEQ ID NO: 137-154) and has substantially the same ability to bind thrombin. Ligand. RNA sequence (SEQ ID NO: 9):
5′-GGAUCGAAG (N) ₂ AGUAGGC-3 ′
55. The RNA ligand of claim 54, comprising: RNA sequence (SEQ ID NO: 10):
5'-GCGGCUUUGGGCGCCGUUGCUU-3 '
The RNA ligand according to claim 6, further comprising:   55. The RNA ligand of claim 54, wherein the ligand is substantially homologous to the ligand of claim 59 and has substantially the same ability to bind thrombin.   55. The RNA ligand of claim 54, wherein the ligand has substantially the same structure as the ligand of claim 59 and has substantially the same ability to bind to thrombin.   55. The RNA ligand of claim 54, wherein the ligand is substantially homologous to the ligand of claim 60 and has substantially the same ability to bind to thrombin.   55. The RNA ligand of claim 54, wherein the ligand has substantially the same structure as the ligand of claim 60 and has substantially the same ability to bind to thrombin.   55. The RNA ligand of claim 54, prepared by the method of claim 49. A method for identifying a nucleic acid ligand for basic fibroblast growth factor (bFGF) comprising:
a) preparing a candidate mixture of RNA nucleic acids;
b) contacting the candidate mixture with bFGF, wherein nucleic acids with increased affinity for bFGF may be fractionated from the remainder of the candidate mixture;
c) fractionating members of the candidate mixture based on affinity to bFGF; and d) amplifying a molecule selected from a candidate mixture having a relatively high affinity for bFGF to be relatively high for bFGF Obtaining a mixture of nucleic acids enriched for sequences having affinity.   68. The method of claim 66, further comprising e) repeating steps b), c) and d).   68. The method of claim 66, wherein the candidate mixture of RNA nucleic acids consists of single stranded nucleic acids.   68. An RNA nucleic acid ligand for bFGF identified by the method of claim 66.   70. The nucleic acid ligand of claim 69, which is a single stranded nucleic acid.   A purified, isolated, non-naturally occurring RNA ligand for bFGF.   72. The RNA ligand of claim 71, wherein the ligand is selected from the group consisting of the sequences shown in Tables II and III (SEQ ID NOs: 28-67).   The ligand is substantially homologous to a ligand selected from the group consisting of the sequences shown in Tables II and III (SEQ ID NO: 28-67) and has substantially the same ability to bind to bFGF. 72. The RNA ligand of claim 71.   72. The RNA ligand of claim 71, wherein the ligand is chemically modified at the ribose and / or phosphate and / or base positions.   72. The ligand has substantially the same structure as the sequences shown in Tables II, III and IV (SEQ ID NO: 28-89) and has substantially the same ability to bind bFGF. The RNA ligand according to 1. RNA sequence (SEQ ID NO: 27):
5'-CUAACCNGG-3 '
72. The RNA ligand of claim 71, comprising: RNA sequence (SEQ ID NO: 43):
5'-RRGGHAACGGYWNNGDCAAGNNNCCYY-3 '
72. The RNA ligand of claim 71, comprising:   72. The RNA ligand of claim 71, wherein the ligand is substantially homologous to the ligand of claim 76 and has substantially the same ability to bind bFGF.   72. The RNA ligand of claim 71, wherein the ligand has substantially the same structure as the ligand of claim 76 and has substantially the same ability to bind bFGF.   72. The RNA ligand of claim 71, wherein the ligand is substantially homologous to the ligand of claim 77 and has substantially the same ability to bind bFGF.   72. The RNA ligand of claim 71, wherein the ligand has substantially the same structure as the ligand of claim 77 and has substantially the same ability to bind bFGF.   72. The RNA ligand of claim 71, wherein the ligand is an inhibitor of bFGF.   72. The RNA ligand of claim 71, wherein the ligand is selected from the group consisting of the sequences shown in Table II (SEQ ID NO: 27-42).

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