WO1993012234A1 - Reactifs antiviraux a base de proteines de liaison de l'arn - Google Patents

Reactifs antiviraux a base de proteines de liaison de l'arn Download PDF

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WO1993012234A1
WO1993012234A1 PCT/US1992/010770 US9210770W WO9312234A1 WO 1993012234 A1 WO1993012234 A1 WO 1993012234A1 US 9210770 W US9210770 W US 9210770W WO 9312234 A1 WO9312234 A1 WO 9312234A1
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polypeptide
rna
seq
hiv
cleavage
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PCT/US1992/010770
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Sumedha D. Jayasena
Brian H. Johnston
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Sri International
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Priority claimed from US07/808,452 external-priority patent/US6063612A/en
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Publication of WO1993012234A1 publication Critical patent/WO1993012234A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3511—Conjugate intercalating or cleaving agent
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to RNA-binding proteins modified to contain nucleic acid cleaving moieties, in particular, viral trans-acting pro ⁇ teins can be used as specific antiviral reagents.
  • the invention further includes methods for genera ⁇ ting said antiviral reagents, methods of cleaving viral nucleic acid, and methods of inactivating viral nucleic acid in cells.
  • phenanthroline attached to an oligonucleotide or polypeptide will bind cupric ion and this complex can be used to cleave DNA.
  • a reducing agent the bound cupric ion is reduced to cuprous ion, which reduces molecular oxygen to produce hydrogen peroxide.
  • the H-jO- reacts with the cuprous complex to form a copper-oxo species that is directly responsible for cleavage.
  • Chen et al. (1987) used this approach to convert the E. coli trp repressor to a site-specific deoxyribonuclease.
  • Copper-phenanthrolene has also been tethered to oligonucleotides to induce sequence-specific cleavage of single-stranded and double-stranded DNA (Francois et al., 1989).
  • An alternative but chemically analogous system utilizes EDTA-chelated iron tethered to an oligonucleotide to cleave DNA.
  • the present invention describes a polypeptide having site-specific RNA binding, where the polypeptide is modified to contain a moiety capable of cleaving an RNA backbone, in particular, viral polypeptides having site- specific viral RNA-binding.
  • exemplary of such polypeptides are the polypeptides presented as SEQ ID N0:1, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:22.
  • a number of cleaving moieties are useful in the practice of the present invention including the following: phenanthroline Cu(II) , Zn(II) , Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
  • the RNA-binding polypeptide can be either the HIV TAT or REV proteins, or polypeptides derived therefrom.
  • a preferred embodiment of the present invention is the polypeptide having the sequence presented as SEQ ID N0:1, which further contains an end-terminal cysteine residue, and where the cleaving moiety is phenanthroline Cu(II) .
  • Another preferred embodiment is the polypeptide having the sequence presented as SEQ ID NO:2 where the cleaving moiety is phenanthroline Cu(II) .
  • a further embodiment of the polypeptide cleaving reagents of the present invention is the generation of fusion polypeptides containing the RNA-binding polypeptide coding sequence fused in frame to a non-specific nuclease.
  • the non-specific nuclease may be covalently bound to the RNA-binding polypeptide.
  • One preferred embodiment of this aspect of the present invention is the polypeptide having the sequence presented as SEQ ID NO:2 coupled to Staphylococcal non ⁇ specific nuclease.
  • the present invention also includes a method of cleaving a target RNA. The method involves contacting an RNA molecule containing a cognate RNA-binding site with the RNA-binding polypeptide cleaving reagent. Both chemical and nuclease cleaving reagents are useful in this aspect of the present invention.
  • the RNA molecule is an HIV RNA and the RNA-binding polypeptide is an HIV- encoded RNA-binding protein, such as TAT, REV, or polypeptides derived therefrom.
  • the RNA-binding polypeptide reagent is supplied at a concentration effective to produce cleavage of the target RNA molecule.
  • Cleavage reactions may further include the addition of a reducing agent, such as mercaptopropionic acid, N-acetyl cysteine, or ascorbate.
  • the cleavage reactions include the addition of the polypeptide reagent, CuS0 4 and mecaptopropionic acid.
  • the invention further includes a method of inhibiting expression of viral antigens in infected cells.
  • the method in ⁇ volves exposing the infected cells to the above described polypeptide cleaving reagent where the polypeptide reagent binds, site-specifically, to the RNA target and is able to be taken up by the infected cells.
  • the cells are exposed to a concentration of the polypeptide reagent which is effective to produce reduction in (i) viral antigen expression or (ii) viral transcription in the infected cells.
  • the cells may be exposed to the polypeptide reagent in the presence of a reducing agent, such as N-acetyl cysteine and ascorbate.
  • the viral RNA is HIV RNA and the polypeptide reagent is based on the HIV-encoded TAT or REV proteins, or polypeptides derived therefrom.
  • the polypeptide reagent includes a non-specific nuclease, expression of the polypeptide reagent in infected and/or un-infected cells provides a useful gene therapy to fight viral disease.
  • the cleaving agents of the present invention are oligonucleotides having nuclease resistant backbones to which a moiety capable of cleaving RNA backbones has been attached.
  • Figure 1 illustrates the sequence and structure of a -variety of TAR elements.
  • Figure 1A shows the TAR element of HIV-1 (also presented as SEQ ID NO:3) .
  • Figure IB shows a TAR element of HIV-2 (also presented as SEQ ID NO:4) .
  • Figure 1C shows a truncated HIV-1 TAR element designated ⁇ TAR (also presented as SEQ ID NO:5).
  • Figure ID illustrates the binding of a TAT-peptide, having an attached cleaving moiety, to a TAR element- containing target RNA.
  • Figure 2A presents the primary coding sequence of the HIV-1 TAT protein. This sequence is also presented as SEQ ID N0:1. The region in bold represents the nuclear targeting domain.
  • the underlined polypeptide a proteolytic product of wild type TAT protein, binds specifically to TAR- element-containing RNA (Weeks et al. , 1990) .
  • Figure 2B presents the sequence of the TAT-derived polypeptide, TAT24C (SEQ ID NO:2), which is derived from the underlined sequence of Figure 2A.
  • Figure 2C illustrates a phenanthroline moiety attached to a cysteine residue of a polypeptide.
  • Figures 3A-D show the results of gel shift mobility assays using modified and unmodified polypeptides and different RNA substrates.
  • Figure 4 presents the sequence of the HIV-1 encoded REV protein. This sequence is also presented as SEQ ID NO:6.
  • Figure 5 illustrates the chemistry of the at- tachment of a phenanthroline moiety to a cysteine- containing polypeptide.
  • Figure 6 illustrates the use of 2-iminothiolane for attachment of cleaving moieties to amino groups of polypeptides.
  • Figure 7 shows one scheme for attachment of iminodiacetic acid to polypeptides for Zn(II) binding.
  • Figures 8A and 8B show the results of cleavage assays using a variety of cleaving agents and an HIV-1 TAR-element containing substrate.
  • the drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
  • Figure 8C shows the results of cleavage assays using a variety of cleaving agents and a truncated HIV-1 TAR-element-containing substrate.
  • the drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
  • Figure 9A shows the results of cleavage assays using a variety of cleaving agents and an HIV-2 TAR-element containing substrate.
  • the drawing of the RNA substrate indicates cleavage sites (arrows) of TAT-based cleaving agents.
  • Figure 9B shows the cleavage products resulting from using tRNA as a substrate RNA.
  • Figure 10 provides an overview of a method for targeting and inactivation of HIV mRNA using oligonucleotides which contain cleaving moieties.
  • Figure 11 shows, in bold, potential triplex target sites within the HIV-1-LTR region: A (SEQ ID NO:8), B (SEQ ID NO:9), and C (SEQ ID NO:10).
  • Figure 12 shows oligonucleotides [A 1 (SEQ ID N0:11) and 2 (SEQ ID NO:12), B 1 (SEQ ID NO:13) and 2 (SEQ ID NO:14), and C 1 (SEQ ID NO:15) and 2 (SEQ ID NO:16)] designed to target the sequences presented in Figure 11 A, B, and C, respectively.
  • Next to each of these oligonucleotides is the general pattern of base triplets expected when triplexes are formed.
  • Oligonucleotides K (SEQ ID N0:17), L (SEQ ID N0:18), and M (SEQ ID N0:19) are the control oligonucleotides.
  • Figure 13 shows a schematic representation of triplex formation at an mRNA target site (bold) using a linear complementary oligonucleotide where the end loop contains basic oligonucleotides (-X- ) .
  • the RNA sequence is presented as SEQ ID NO:21 and the oligonucleotide is presented as SEQ ID NO:20, where N is an basic residue.
  • Figure 14 shows a schematic representation of the HIV-1 REV response element (RRE) ; this sequence is also presented as SEQ ID NO:23.
  • SEQ ID NO:24 presents the sequence of a truncated RRE binding site which corresponds to Stem II in the figure.
  • RNA-specific binding proteins can be adapted to perform site-specific cleavage of a target RNA when the target RNA contains the cognate binding site to which the RNA-binding protein binds.
  • One important application of these protein-based cleaving agents is the inactivation of mammalian viruses; in particular, RNA binding proteins modified to accomplish site specific cleavage can be used for inactivating RNA viruses, including the human immunodeficiency viruses (HIV) .
  • HIV human immunodeficiency viruses
  • RNA target site specific recognition RNA target site specific recognition
  • known or identifiable recognition sequence the ability to get the protein or polypeptide into cells containing the target RNA
  • a relatively small protein binding domain is preferable
  • fragments containing the protein binding domain should compete well for binding at the RNA target site with the native protein from which they are derived.
  • RNA-binding proteins Two examples of RNA-binding proteins useful in the practice of the present invention are the TAT and REV proteins encoded by HIV-l.
  • TAT consists of 86 amino acids and is a potent transactivator of long terminal repeat (LTR)-directed viral gene expression and is essential for viral replication (Dayton et al., 1986; Fisher et al., 1986).
  • LTR long terminal repeat
  • the amino acid sequence of the TAT protein is presented as SEQ ID NO:l.
  • TAT-induced transactivation requires the presence of the TAR (transactivation response) element, located at the untranslated 5' end of the viral mRNA element.
  • the RNA sequence of the TAR element is presented as SEQ ID NO:3.
  • the TAR element is capable of forming a stable stem-loop structure (Muesing et al., 1987) in the native viral RNA ( Figure 1) .
  • a 3 nucleotide (nt) bulge on the stem of TAR has been demonstrated to be essential for specific and high-affinity binding of the TAT protein to the TAR element (Roy et al., 1990; Cordingley et al . , 1990; Dingwall et al., 1989; Weeks et al., 1990).
  • the ability of (i) purified TAT protein and (ii) polypeptide fragments derived from TAT, which contain the nuclear targeting domain, to bind in vitro to RNA containing the TAR element make TAT a useful model for the RNA-cleaving reagents of the present invention.
  • the integrity of the stem and the initial U22 of the bulge are important or TAT protein binding (Roy et al. , 1990b) .
  • TAT protein binding Rost al. , 1990b
  • other sequences that do not affect the binding of the TAT protein to the TAR site are needed for trans-activation of tran ⁇ scription.
  • One such region is the sequence at the loop, which is required for the binding of cellular factors that may interact with the TAT protein to mediate transactivation (Gatignol et al. , 1989; Gaynor et al. , 1989; Marciniak et al. , 1990a; Gatignol et al., 1991).
  • the important components of the protein/RNA interaction are (i) the portion of the protein involved in binding to the specific target sequence, and (ii) the RNA target sequence required for binding.
  • TAT protein both full- length TAT (86 amino acids) and a truncated TAT protein (the N-terminal 72 amino acids) have been expressed as fusion proteins in Escherichia coli (Weeks et al., 1990).
  • the TAT proteins were linked to the E . coli catabolite activator protein via a protease recognition site.
  • polypeptide fragments were recovered from each fused protein which resulted from an unexpected cleavage between Gly48 and Arg49 ( Figure 2) , at the start of the lysine/arginine-rich RNA-binding region.
  • the fragments resulting from this cleavage, TRF38 and TRF24 corn-prise the regions extending from Arg49 to residue 86 and Arg49 to residue 72, respectively.
  • Both of these polypeptide fragments specifically bound RNA containing the TAR site (TAR-RNA) .
  • a synthetic 14-residue polypeptide spanning the basic region (amino acid residues 48-61 of the TAT protein) bound TAR-RNA as well.
  • TRF38 Up to three copies of TRF38 could bind to the wild- type TAR (wt-TAR) site, the first with an apparent dissociation constant of 5 nM and a second copy with an apparent dissociation constant of 20 nM. Only one copy of the TRF38 polypeptide could bind to a truncated version of TAR consisting of the minimal TAT binding site: the minimal site consists of 26 bases containing the upper part of the wt-TAR ( Figure 1C; Muesing et al., 1987). Similar binding characteristics were found for TRF24.
  • TAT24C A polypeptide similar to TRF24, designated TAT24C, was chemically synthesized (Example 1A) .
  • TAT24C consists of amino acid residues 49-72 of the TAT protein (Figure 2A) and an additional cysteine residue at the C-terminus ( Figure 2B, SEQ ID NO:2).
  • the TAT24C polypeptide was purified by HPLC and reacted with 5-iodoacetamido-l,10- phenanthroline to attach a 1,10-phenanthroline moiety to the cysteine residue (peptide designated TAT24C-phen, see below) .
  • RNA substrates are prepared as described in Example 2.
  • Several TAR-containing RNAs were synthesized to use as substrates in binding assays to test the binding activity of the modified polypeptide
  • TAT24C-phen (Example 2) .
  • the predicted secondary structures of the target RNAs are shown in Figure 1C.
  • the RNA substrate designated HIV-1 TAR is the 57-nt RNA stem-loop structure found in native HIV-1 mRNA (Sharp et al. , 1989).
  • the RNA substrate designated ⁇ TAR is a truncated RNA containing the minimum TAT binding site (nt 17-43) (Weeks et al., 1990).
  • HIV-1 the etiologically associated virus of AIDS, another retrovirus, termed type 2 (HIV-2) has been reported (Clavel et al., 1986). HIV-2 also possess a functional TAT gene (Arya et al.
  • HIV-2 TAT responsive element Two subelements responsible for the TAT-mediated transactivation in the HIV-2 TAT responsive element (TAR) have been identified and contain two stem-loop struc ⁇ tures confined to +1 - +103 nucleotides (Arya et al., 1985).
  • the sequence of HIV-2 TAR RNA used in the present study is from +13 - 90 nucleotides and contains both stem-loop structures.
  • FIG. 3 shows a photograph of a representative gel mobility shift assay.
  • discrete bands having retarded mobility for samples containing either phenanthroline- odified or un ⁇ modified polypeptides demonstrate the binding of both polypeptides to each of the three RNAs con ⁇ taining TAR elements ( Figure 3A-C) .
  • the attach- ment of the extra cysteine and the phenanthroline moiety at the C-terminus of the TAT24 polypeptide does not substantially affect binding to the TAR site.
  • Essentially no retardation of tRNA was observed when it was incubated with high levels of the TAT24C polypeptide ( Figure 3D) , indicating relatively specific binding to TAR-site-containing RNA substrates.
  • TAT protein As well as fragments of TAT containing the nuclear targeting region, are rapidly taken up by cells.
  • the TAT protein taken up by cells in this fashion specifically activates HIV-1 LTR-linked gene expression (Green et al., 1988; Frankel et al., 1988).
  • the entry of polypeptides containing (i) the nuclear targeting region of TAT and (ii) nucleic acid cleaving agents can be evaluated as described below.
  • REV protein is a regulatory factor essential for viral replication; it is required for the production of viral structural proteins. It appears to exert its effect at the level of splicing and perhaps transport of viral mRNA into the cytoplasm (Malim et al., 1989a, 1989b; Felber et al., 1989); further REV appears to increases the stability of unspliced HIV mRNA (Felber et al., 1989).
  • the REV protein consists of 116 amino acids ( Figure 4, SEQ ID NO:6), encoded by two exons.
  • arginine-rich domain acts as the nuclear tar ⁇ geting domain (Malim et al., 1989b). Mutational analysis has demonstrated that some C-terminal deletion mutants of the REV protein are non ⁇ functional, in terms of normal REV functions, but are trans-dominant as illustrated by competitive inhibition of wild-type REV functions.
  • REV response element The action of REV requires the presence of a target sequence termed the REV response element
  • RRE Figure 14, SEQ ID NO:23
  • Malim et al. , 1989a, 1989b RRE
  • RRE has been mapped to a 234-nucleotide region capable of forming four stem-loop structures and one branched stem-loop structure
  • REV offers another potential means of targeting a cleaving agent specifically to HIV RNA and has a potential advantage over the TAT protein in that REV has more potential binding sites.
  • RNA-binding proteins and their cognate binding sites can be characterized as has been de ⁇ scribed above for the TAT protein and TAR site. Truncated versions of any such protein and/or binding site can be evaluated for protein/-peptide binding using, for example, the gel mobility shift assay or standard filter binding assays (Sauer et al. ; Radding et al.). Further, cellular uptake can be evaluated. If the protein is not adequately taken up by cells at concentrations useful to provide intracellular catalytic function, i.e., cleavage, then alternative methods of cellular uptake, such as targeted liposomal delivery where the liposomes carry target cell surface recognition moieties, can be employed to get polypeptide fragments into target cells.
  • a number of chemical moieties are capable of cleaving nucleic acid substrates including phenan ⁇ throline (Chen et al., 1986, 1987; Francois et al., 1989; Ebright et al., 1990), Fe(II)-EDTA (Dreyer et al., 1985; Dervan, 1986; Moser et al., 1987; Maher et al., 1989; Sluka et al. , 1987), Cu(II)-bipyridine, Cu(II)-terpyridine, and Zn(II) (Modak et al., 1991; Eichhorn et al., 1971; Ikenaga et al., 1974; Breslow et al., 1989).
  • These chemical cleaving moieties can be employed in the present invention as exemplified below with reference to the TAT24 C-phen protein.
  • Nucleic acid cleaving moieties are attached to the TAT derived polypeptides as described in Example IB. The chemically synthesized
  • HPLC-purified polypeptides are reacted with 5-iodoacetamido-l,10-phenanthroline (phenanthroline moiety) to obtain polypeptides containing the phenanthroline moiety uniquely attached to the side chain of the cysteine residue.
  • Figure 5 illustrates the attachment of a phenanthroline moiety to cysteine-containing polypeptides.
  • TAT24-C polypeptide (SEQ ID NO:2), consisting of that 24-residue domain plus a single cysteine residue at the C-terminus, was chemically synthesized (Example 1A) .
  • a phenanthroline moiety was then attached to the sulfhydryl group of the cysteine to obtain TAT24C-phen (Example IB) .
  • the cleaving agent can also be attached at residues other than cysteines.
  • phenanthroline can be attached to the side chain- amino groups of lysines and arginines, as well as to the amino group at the N-terminus, by reacting the protein first with 2-iminothiolane hydro ⁇ chloride, followed by coupling with 5-iodoace- tamido-l,10-phenanthroline (Chen et al. , 1987). This coupling is illustrated in Figure 6 and described in Example IB. Because of the higher loading of cleaving moieties per protein/peptide molecules, such protein/peptide molecules are expected to be efficient reagents for cleavage and are expected to be resistant to in vivo degradation.
  • nucleic acid cleaving agents can also be used in the methods of the present invention, including Fe(II)-EDTA,
  • the intracellular reduction potential can be affected using N-acetyl cysteine, which increases the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991), in order to assist in keeping the metal atom of the cleaving agent in the reduced state.
  • N-acetyl cysteine increases the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991)
  • RNA degradation is known to be induced by divalent metal ions, especially Zn(II) , in the absence of a reducing agent.
  • the reduced hydrolytic cleavage compared to reaction in the presence of a reducing agent can be at least partly of set by having more than one chelating molecule attached to the polypeptide.
  • One technique for tethering Zn(II) to a protein is via coordination by iminodiacetic acid (Aldrich, Milwaukee WI) : one possible scheme for such attachment is shown in Figure 7. Briefly, iminodiacetic acid is converted to its diethyl ester to protect carboxylic function ⁇ alities. The resulting product is condensed with iodoacetic acid in the presence of dicyclohexyl carbodiimide (DCC) to obtain compound 3 ( Figure 7) . Compound 3 is then reacted with, for example, a polypeptide containing a cysteine residue.
  • DCC dicyclohexyl carbodiimide
  • the polypeptides are screened for specific binding to their cognate nucleic acid binding site, the polypeptides carrying nucleic acid cleaving moieties are next evaluated for their ability to cleave the target nucleic acid.
  • a non-specific enzymatic nuclease can be attached to a sequence specific RNA binding protein to form a sequence-specific ribonuclease.
  • Staphylococcal nuclease a non-specific nuclease that attacks both RNA and DNA, has been converted to site- specific DNA endonuclease by attaching the protein to an oligonucleotide (Corey et al., 1987; Pei et al., 1990).
  • hybrid fusion pro ⁇ teins are generated between the RNA sequence- specific protein and the coding sequence of Staphylococcal nuclease.
  • the hybrid proteins can be expressed using any number of standard expres ⁇ sion systems (e.g., "CLONTECH” commercially available vectors) .
  • CLONTECH commercially available vectors
  • the hybrid protein is expressed in E. coli using the OmpA-derived expression system (plasmid pONFl) already adopted for the secretion of staphylococcal nuclease (Takahara et al. , 1985) .
  • the signal peptide required for the secretion of o pA protein is fused to the nuclease gene to obtain large amounts of secreted nuclease: the nuclease is then released from the signal polypeptide by appropriate processing.
  • DNA sequences encoding the TAT, REV, or derivative RNA-binding poly ⁇ peptides are cloned adjacent to and in-frame with the staphylococcal nuclease gene in plasmid pONFl.
  • the RNA-binding protein coding sequence can be generated by any number of methods including polymerase chain reaction amplification (Mullis et al. ; Mullis) and standard cloning technology (Ausubel et al.
  • RNA-binding protein/- peptide coding sequence is performed by standard cloning methods (Ausubel et al. ; Maniatis et al.; Sambrook, et al.) .
  • the resulting hybrid proteins are then expressed in E. coli and purified (Takahara et al., 1985).
  • hybrid proteins/peptide nucleases to bind their cognate nucleic acid substrate is evaluated as described above using, for example, the gel mobility shift or filter binding assays.
  • hybrid protein/peptide nucleases are screened for specific binding to their cognate nucleic acid binding site, they are next evaluated for their ability to cleave the target nucleic acid.
  • the chemical nuclease activity of Cu(II)-com- plexed l,10-phenanthroline derives from an oxida- tive attack on the sugar ring by a copper-oxo species generated in the presence of a reducing agent (Sigman et al., 1990).
  • RNA target molecules containing the binding sites were 5'-end- labeled, purified on denaturing polyacryl- amide gels, and annealed for use as substrates for cleavage (Example 2) .
  • a typical cleavage reaction contained 10 3 cpm of end-labeled RNA and 40-100 ng of polypeptide.
  • Example 4A describes RNA cleavage reactions utilizing the TAT24C-phen polypeptide. The target RNA was incubated with TAT24C-phen at 25"C for 10 min before the cleavage was initiated by adding CuS0 4 and mercaptopropionic acid.
  • Cu(II) is different from their reactivity toward the polypeptide.
  • the effect of the TAT24C-phen was examined relative to the HIV-2 TAR site (Example 4C) .
  • the primary cleavage site found on the HIV-2 TAR is somewhat unexpected ( Figure 9, lanes 1 and 2).
  • the cleavage site of the HIV-2 target was anticipated to be predominantly at the loop close to the TAT binding site (loop 1, Figure 9A) .
  • the TAT24C-phen polypeptide appears to cleave the HIV-2 TAR-RNA at a site located towards the 5' end of its binding site ( Figure 9A) .
  • the primary cleavage site in the HIV-2 TAR site is not in the loop and does not appear to have unpaired bases (as implied by the SI nuclease cleavage reactions the results of which are shown in lane 4, Figure 9A) .
  • the primary site of cleavage in the HIV-2 substrate is likely the consequence of the tertiary structure of the HIV-2 TAR RNA.
  • the HIV-2 target consists of two stem- loop structures: the HIV-2 TAR RNA sequence may have a complex tertiary structure reduces the otherwise favorable interaction of the polypeptide-bound cleaving moiety with the loop.
  • the TAT24C-phen polypeptide does, however, cleave both HIV-2 target loops.
  • the cleavage of the loop 1 is ex ⁇ pected and the cleavage of the remote second loop may be the result of the two loops being in a spatially close orientation within the overall secondary structure of the HIV-2 substrate.
  • cleavage sites of the RNA tar ⁇ gets lie on either side of the bulge where the TAT protein is known to bind (Roy et al., 1990;
  • HIV-2 TAR In contrast to cleavage of HIV-1 TAR, for which the primary site is at the loop adjacent to the TAT binding site, cleavage of HIV-2 TAR takes place mainly at the stem, roughly midway between the two loops ( Figure 9A, lanes 1 and 2) .
  • HIV-2 TAR has two 2-nt bulges, both of which have the consensus TAT binding motif (Weeks et al. , 1990; Green- et al., 1988; Frankel et al. , 1988; Milligan et al., 1987; Arya et al. , 1988; Weeks et al., 1991; Murakawa et al. , 1989).
  • RNA target mole ⁇ cules using modified binding polypeptides, i.e., proteins known to have binding sites in a selected RNA target molecule.
  • modified binding polypeptides i.e., proteins known to have binding sites in a selected RNA target molecule.
  • REV-derived polypeptides encompassing the nuclear targeting domain are synthesized and attached to cleaving agents as described for reagents based on the TAT protein.
  • a synthetic peptide spanning the basic domain of the REV protein (SEQ ID NO:22) has been shown to bind specifically to the RRE target (Kje s et al., 1991) ; in vitro the same peptide inhibits the splicing of mRNA containing RRE.
  • target RNA molecules can be cleaved using polypeptides modi ⁇ fied with iminodiacetic acid in the presence of ZnCl 2 .
  • RNA binding proteins or polypeptides which are derived from these proteins, depends on seve ⁇ ral factors: (a) the binding affinity and speci- ficity of the reagent; (b) the spatial positioning of the cleaving moiety; (c) the nature of the cleaving reagent; and (d) reaction conditions.
  • the specificity and the binding affinity of a binding polypeptide to its cognate binding site in an RNA target molecule may potentially be increased by increasing the length of the polypeptide, for example: (i) by extending the polypeptide length from the N-terminus, (ii) the C-terminus, or (iii) both ends of the basic nuclear targeting domain of a transactivator.
  • TAT24C-phen In the case of the TAT and REV proteins the maximum number of residues needed to maximize specific binding is not expected to be more than 50 residues, because both trans-activators are relatively small, on the order of 100 residues, and both have functional domains besides the RNA binding domain.
  • Two other approaches to increa ⁇ sing the binding specificity of TAT24C-phen to the target TAR site are as follows: (i) evaluating in vitro generated mutations (Ausubel et al.) for mutations that increase the binding specificity of the protein/peptide to the cognate binding site (Sauer et al.); (ii) chemical alterations of the polypeptides which can effect a general improve ⁇ ment of RNA binding, such as replacing Asp and Glu with Asn and Gin.
  • chemical modifi ⁇ cation can be used to convert Asp and Glu to esters or amides to increase the net positive charge of the polypeptide.
  • Chemical modification reactions that occur in solution can be performed on the polypeptide bound to the target RNA where possible, so that sites critical for binding are protected against alteration (Galas et al. ; Siebenlist et al.).
  • the spatial positioning of the cleaving moi ⁇ ety may be adjusted. Because of the three-dimen ⁇ sional folding of polypeptides, the position of the cleaving agent within the polypeptide molecule can be crucial for the cleavage. In the cases of TAT and REV proteins the three dimensional struc ⁇ ture of the proteins is not known. Accordingly, favorable locations for the placement of the cleaving moiety can be empirically determined; the single cysteine residue can be placed at several positions, including the N-terminus, C-terminus, and internal positions of the polypeptide which do not affect RNA-binding ability.
  • cleaving reagent in order to improve the efficiency of cleavage reactions using RNA binding proteins or polypeptides derived from these proteins the nature of the cleaving reagent can be modified as described above, using chemical or enzymatic cleaving moieties.
  • reaction conditions can be modified to improve cleavage of RNA substrates by, for example, increasing reduction potential in the reaction mixture or intracellularly by, for example, adding N-acetyl cysteine (or perhaps ascorbate) to the system.
  • In vitro reaction conditions can also be modified by altering temperature, ionic conditions, the amount and type of reducing agent, and pH.
  • the cleavage assay will be used to assess the effects of the above factors on the efficiency of the cleavage reaction.
  • Single variables will be modified to evaluate efficacy. For example, to assess the cleavage induced by different peptides, an identical concentration of the different pep ⁇ tides are used in reaction mixtures containing the other reagents at fixed concentrations. After the cleavage reaction has been carried out for a specified period, digested end-labeled RNAs (e.g., HIV-1 TAR) will be resolved on sequencing gels, the gels will be autoradiographed, and bands corresponding to starting material (intact RNA) and cleavage products will be excised.
  • digested end-labeled RNAs e.g., HIV-1 TAR
  • the radioactive counts present in the excised bands will be determined by scintillation counting.
  • the relative concentrations of cleavage product to starting material is then determined ( (cpm prod /cpm Pro +c P in i ntact ) 10 °) •
  • Densitometry scanning can also be used to evaluate efficiency of cleavage reac ⁇ tions by using films which have not been overex ⁇ posed.
  • RNA molecules contain both single- and double-stranded regions that can offer targets for oligonucleotide binding (Zamecnik et al., 1986; Rittner et al., 1991).
  • RNA oligonucleotide agents must con ⁇ tinuously bind to the target molecules in such a way as to inactivate them.
  • a clea ⁇ vage agent is attached to RNA oligonucleotides, the oligonucleotides only need to bind the target RNA long enough to cleave it in order to achieve permanent inactivation.
  • a chosen cleaving group (see above for chemical and enzyma- tic cleaving groups) is attached to oligonucleo ⁇ tides which are resistant to cleavage by endo ⁇ genous nucleases.
  • backbones include deoxy- ribose or ribose sugar moieties connected by methyl phosphonate or phosphorothioate linkages (Miller et al., 1985).
  • RNA-binding oligonucleotides have sequences of the following two types: (i) sequences designed to form a du ⁇ plex with putatively single-stranded regions of a target RNA, and (ii) sequences designed to form triplexes with homopurine regions of the DNA which encodes the RNA-target, for example, a DNA pro- virus.
  • one mRNA target site is the TAR region, because base pairing at this site by a complementary oligonucleotide is expected to block formation of the stem-loop structure required for binding and transactivation by TAT.
  • An inter-molecular duplex is potentially more stable than the intra-molecular stem-loop duplex due to the absence of unpaired bases. Further, such an inter-molecular duplex may be able to displace the stem-loop structure by pairing initially with the loop or with single-stranded regions adjacent to the stem, particularly in view of the observation that alteration of non-essential sequences adjacent to TAR create competing secondary structures which inhibit TAR function (Berkhout et al., 1989).
  • a major advantage of targeting the DNA pro- virus associated with an RNA virus is that typically only one, or a few copies, of integrated, transcriptionally active DNA are present per cell in contrast to many copies of mRNA which may be present in an infected cell (Soma et al., 1988).
  • Homopurine-homopyrimidine regions of duplex DNA can bind single-stranded oligonucleotides having the same sequence as either the homopurine or the homopyrimidine strand of the target DNA but with the reverse polarity (Dervan, 1986) , forming purine-purine-pyrimidine or pyrimidine-purine-pyrimidine triplexes, respectively.
  • the purine-purine-pyrimidine triplexes typically require a divalent cation such as Mg ++ or Zn ++ for their stability but are relatively independent of pH (Lyamichev et al., 1991) .
  • the pyrimidine-purine-pyrimidine triplexes require divalent cation but are favored by slightly acid pH.
  • triple helix approach for targeting DNA to inhibit expression has had limited use due to the requirement for homopurine target sequences.
  • Triplex formation at an oligopurine*oligopyrimi ⁇ dine tract can be induced by a single strand consisting of either only pyrimidines or only purines. Sequence-specific recognition by the oligopyrimidine strand relies on the formation of PyPuPy (C+ «GC and T ⁇ T) base triplets (Moser et al., 1987). In this case, the oligopyrimidine strand is parallel to the purine tract of the duplex.
  • oligopurine strand lies anti-parallel to the purine tract of the duplex, and sequence-specific recognition in this case is brought about by Pu «PuPy (G «GC and A*AT) base triplets (Kohwi et al., 1988; Beal et al.,
  • a sequence of 15-18 purines is required to achieve sufficient specificity, and this requirement limits the triplex approach in controlling the expression of a particular gene. Although long homopurine stretches do occur in viral genomes, finding such a sequence within a gene vital to the virus can be difficult.
  • triplex formation can occur at tandem oligopurineOligopyrimidine sequences using normal DNA, without any unnatural linkages or synthetic base analogues.
  • sequences utilize both types of base triplets, Pu'PuPy and PyPuPy, in forming a triplex.
  • this approach allows the formation of triplexes at base sequences made up of both purines and pyrimidines.
  • the incorporation of Pu «PuPy base triplets has the advantage that triplex formation does not demand low pH, which is usually the case when the C+ «GC base triplet is involved.
  • triplex- forming oligonucleotides are designed to interact with ho opurine-homopyrimidine sequences in the pro-virus.
  • three potential targeting sites are three potential targeting sites (sequences in bold, Figure 11A, 11B, 11C) for targeting with single-stranded oligonucleotides.
  • These cleaving-oligonucleotide reagents bind and cleave the DNA provirus as well as the mRNA of HIV, increasing the likelihood of preventing viral replication.
  • All three target sites are located in the control region of the HIV-LTR (i.e., upstream of the transcription initiation site) and therefore do not interact with mRNA sequences to function as anti-sense mediators.
  • the potential target sites A, B, and C have different triplex forming motifs:
  • Site A consisting exclusively of purines, is targeted for triplex formation using oligonucleotides A-l and A-2 ( Figure 12) , which are capable of forming triplexes with Pu «PuPy and PyPuPy base triplets, respectively.
  • Site B consists of a tract of pyrimidine residues flanked by two purine tracts and is targeted with oligonucleotides B-1 and B-2 ( Figure 12) , having the correct polarity to bind with two strands of the target (see above) .
  • Site C has some pyrimidines scattered within a highly purine-rich sequence, and oligonu ⁇ cleotides C-l and C-2 ( Figure 12) are directed toward site C.
  • oligonucleotides K, L, and M correspond, respectively, to sites A, B, and C in the reverse polarity and are therefore blocked from triplex formation; these oligonucleotides are used as controls.
  • Test oligomers with and without phenanthroline are used to assess the effect of cleavage. Attachment of 1,10-phenanthroline to oligo ⁇ nucleotides is achieved as follows. During chemi ⁇ cal synthesis each oligonucleotide is synthesized with a thiol group at the 5' end by use of the "C6-thiol modifierTM 11 reagent from Clontech (Palo Alto, CA) according to the manufacturers instruc ⁇ tions.
  • the oligonucleotides are de-protected with NH 4 OH and treated with silver nitrate to expose the thiol group.
  • the oligonucleotide is immediately reacted with 5-iodoacetamido 1,10-phenanthroline as described above to covalently link 1,10-phenan ⁇ throline to polypeptides.
  • pHIV-lLTR-CAT is linearized with Hindlll, end-labeled with 32 P ⁇ 7 ⁇ ATP using polynucleotide kinase, and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment containing the duplex target sequence.
  • this DNA fragment is mixed with an appropriate modified oligonucleotide in a buffer containing 10 mM Tris-HCl, 100 mM NaCl, 100 ⁇ M spermine, and 10 mM MgCl 2 .
  • the pH of the buffer is adjusted depending on the sequence of the target (a lower pH is used for the formation of C+ «GC base triplets) .
  • cleavage is initiated by adding CuSo 4 (to 10 ⁇ M) and mercaptopropionic acid (to 2.5 mM) .
  • Cleavage products are resolved on sequencing gels along with the products of sequencing reactions. This method allows the mapping of the site of triplex formation and the cleavage efficiency (detected by counting the radioactivity of excised gel bands) ; cleavage efficiency is used to quantitate the efficiency of triplex formation.
  • the CAT gene is transiently expressed under the direction of HIV-1 LTR in HeLa cells.
  • HeLa cells are trans- fected with pHIV-lLTR-CAT, using the DEAE-dextran technique (Queen et al., 1983). Twelve hours after transfection, the cells are incubated with an oligonucleotide, as described by Postel et al. (1991) , and itomycin C (SIGMA) is added to the medium to induce CAT expression.
  • SIGMA itomycin C
  • Cells are har ⁇ vested at 12 and 24 hr and CAT activities deter ⁇ mined as described by Gorman et al. (1982) and compared to controls, i.e., cells that have been exposed to control oligonucleotides (K, L, and M) and cells without oligonucleotide treatment.
  • oligonucleotides carrying phenanthroline are complexed with CuS0 4 before being introduced to the cell medium.
  • Ascorbic acid or mercaptopropionic acid
  • Ascorbic acid is supplied to the medium 12 hr after the oligonucleotide treatment and cells are harvested and assayed for CAT activity after another 24 hr.
  • the effect of oligonucleotides in the pre- sence of TAT protein is assayed using p-HIV-lLTR- CAT under stable expression conditions (described above) .
  • CAT-active clones are transfected with a TAT expression vector and these cells, which are transiently expressing TAT are used for oligonu- cleotide treatment followed by the measurement of CAT activity.
  • triplex helix methods An alternative to the above described triplex helix methods is to use an oligonucleotide-based approach where a single-stranded oligonucleotide is capable of forming a triplex with HIV mRNA by contributing two "strands" connected by a hairpin loop ( Figure 10; Figure 13) .
  • This triplex- directed anti-sense approach is expected to be more effective in arresting biological processes such as translation and reverse transcription than is the convention anti-sense approach where a DNA- RNA duplex is formed.
  • Triplex formation in this fashion is highly selective and of high affinity and may not be a substrate for enzymes such as helicases. The action of such helicases has been a potential problem in the conventional anti-sense approach (Bass et al. , 1987).
  • oligonucleotides For cleavage of target RNA substrates, oligonucleotides have a chemical cleaving group attached to one end ( Figure 10) and an inter- calator linked to the other end. Because de- protection procedures are different and indepen ⁇ dent from each other, derivatization at the two ends can be performed at two stages of oligonu ⁇ cleotide synthesis.
  • the cleaving reagents of the present inven ⁇ tion provide means for a method of cleaving RNA targets at specific sites. Such cleavage is useful for the analysis of RNA structure and function as well as diagnostic analyses.
  • One example of a diagnostic application is to isolate RNA from a cell infected with a particular RNA virus. Total or poly-A+ RNA (Ausubel et al.) is end labeled. The RNA is then isolated away from free label and the amount of incorporated label estimated, for example, by scintillation counting.
  • RNA cleaving agent such as an RNA-binding protein combined with a chemical cleaving moiety
  • the cleaving reagents of the present invention are particularly desirable for use with RNA virus tar ⁇ gets or their pro-viral DNA forms: for example, cleaving HIV genomic RNA or pro-viral DNA.
  • the cleaving reagents of the present inven ⁇ tion are also useful in a method of inhibiting expression of RNA viral (e.g., HIV) antigens in cells infected with the virus.
  • the infected cells are exposed to an RNA binding protein or polypeptide modified to contain a cleaving moiety (i.e., the reagent), at a re ⁇ agent concentration effective to produce reduction in viral antigen expression in the infected cells.
  • a cleaving moiety i.e., the reagent
  • both modified and unmodified polypeptides are assayed for their ability to enter the cell.
  • One method to evaluate cellular uptake is to label the poly ⁇ peptides with a fluorescent dye, such as fluores- cein isothiocyanate (FITC) (Pierce, Rockford, IL) at the single cysteine residue.
  • FITC fluores- cein isothiocyanate
  • the fluores ⁇ cent-labeled polypeptides are added to the cell culture medium and the cellular distribution analyzed by fluorescence microscopy.
  • fluorescent labeling is carried out at a single cysteine residue before reacting amino groups with 2-iminothilane for attachment of the cleaving moiety.
  • uptake of the re ⁇ agent polypeptide can be evaluated using radio ⁇ active label since any polypeptide can be easily made radioactive during synthesis (Chen et al. , 1986) .
  • Another alternative is to perform an immuno-fluorescence assay on fixed cells after incubation with the reagent using rabbit anti- peptide- antibodies and rhodamine-conjugated goat anti-rabbit antibodies (Malim et al., 1989).
  • RNA-binding protein can be applied to any protein or polypeptide under investigation, e.g., TAT or REV.
  • TAT covalently attached to the chemical cleaving group, 1,10-phenanthroline results in cleavage of target TAR sequences consistent with polypeptide binding to the 3-nt bulge.
  • RNA-cleaving protein/peptide reagents for example, the in vivo usefulness of the TAT24C-phen polypeptide is tested using a number of cell systems including the following:
  • Chloramphenicol acetyltransferase (CAT) assays HIV-1 LTR-directed CAT activity is mea ⁇ sured under transient expression as well as stable expression conditions.
  • HeLa cells will be transfected with an expression vector containing the entire U3 region and 78 base pairs of the R region of the HIV LTR (e.g., pHIV- 1LTR-CAT (S. Miller, SRI International, Menlo Park CA) ; or Gendelman et al., 1985).
  • the LTR region contains the enhancer, promoter and TAR elements. Transfection is performed using the DEAE-dextran technique (Queen et al. , 1983).
  • the cells are incubated with the polypeptide reagent, over a range of polypeptide concentrations. Mitomycin C is added to the medium to induce CAT expression. Since the HIV-1 LTR is under the influence of NF- kB, the expression of CAT activity can be induced by treating with either UV or mitomycin C (Nabel et al., 1987). After 12 and 24 hours the cells are harvested and CAT activities are determined as described by Gorman et al. (1982) . CAT activities are compared between (i) cell samples which were not treated with the polypeptide reagent, and (ii) cells samples which were treated with the poly ⁇ peptide reagent.
  • Cleavage of the target substrate by the polypeptide reagent is expected to result in a decrease of CAT activity.
  • Polypeptide re ⁇ agents containing phenanthrolene are complexed with CuS0 4 before addition to the cell samples. If the cellular reduction potential is not sufficient for the cleavage to occur, ascorbic acid (or mercaptopropionic acid) is added to the medium.
  • CAT expression system To assess the activity of polypeptide reagents in the presence of wild-type TAT protein, a stable CAT expression system is used. HeLa cells are cotransfected with a 1:5 ratio of pSV2neo (a mammalian integration plasmid which confers neomycin-resistance; Southern et al., 1982) and pHIV-lLTR-CAT plasmids using DEAE- dextran procedure. Cells are selected for G418 resistance, and individual colonies are picked, expanded, and tested for CAT expression. CAT- active clones are transfected with a wild-type TAT expression vector (e.g., pcDEBtat, S. Miller, SRI International; or pAR, available from the AIDS Re ⁇ search and Reference Program) . Cells now expressing TAT transiently are used for polypeptide treatment followed by the measurement of CAT activity.
  • pcDEBtat a mammalian integration plasmid which confers
  • the TAT24C-phen polypeptide reagent is added to the culture media over a range of concentra- tions.
  • the ability of TAT24C-phen to block the transactivation by endogenous TAT protein is determined by measuring chloramphenicol acetyl transferase (CAT) activity over time after the addition of the TAT24C-phen polypeptide.
  • CAT chloramphenicol acetyl transferase
  • HIV antigen levels including p24, asso ⁇ ciated with HIV-infected cells (e.g., by ELISA (Wang et al. , 1988, 1989; Crowe et al., 1990);
  • TAT24C-phen Blocking acute infection — The abil- ity of TAT24C-phen to prevent acute infection by HIV of the following cells will be assessed: PHA-stimulated human peripheral blood lymphocytes, MT4 and Jurkat cells (both CD4+ lymphocyte cell lines) , macrophages, and monocytes infected by monocytropic HIV isolates.
  • TAT24C-phen polypeptide is evaluated using, for example, killing of Jurkat cells. Also, mutagenicity is evaluated with a standard Ames test.
  • the intracellular reduction potential can be modulated using N-acetyl cysteine, which increases the intracellular glutathione level (Roederer et al., 1990; Kalebic et al., 1991).
  • N-acetyl cysteine increases the intracellular glutathione level
  • Such manipulation of the intracellular reduction potential assist in keeping, for example, a copper atom of a cleaving agent in the reduced state.
  • RNA cleaving reagents composed of an RNA- binding protein and a non-specific nuclease also have important in vivo applications. A specific RNA cleaving-hybrid nuclease can be evaluated as described above when the hybrid nuclease is taken up into cells.
  • CAT expression in Hela cells harboring target RNA-CAT fused genes are assayed in the presence and absence of hybrid- nuclease expressed from an independent promoter.
  • the gene for staphylococcal nuclease is cloned adjacent to the TAT or REV gene in plasmids pSV2TAT 72 (or pgTAT) and pCREV, respectively.
  • the resultant plasmids encoding hybrid proteins are transfected into Hela cells carrying either pHIV-CAT or pHIV-env depending on the type of hybrid nuclease.
  • the biological effects of the in vivo expression of TAT24C-nuclease is evaluated using the CAT assay as described above.
  • the effect of the hybrid nuclease containing the gene product of REV will be assayed in Hela cells by quantitating the repression of the production of viral envelope protein as assayed using antibodies against envelope proteins.
  • attaching staphylococcal nuclease to an RNA binding polypeptide e.g., based on TAT and REV
  • a short tether of several amino acids may generate a sequence-specific ribonuclease.
  • Constitutive expression of such an RNA-specific nuclease in an un-infected cell, contained in a population of cells infected with an RNA virus that contains the target RNA binding sequence may confer resistance of the un-infected cells against viral infection.
  • peripheral blood mononucleocyte cells are isolated from the blood of an HIV-positive patient.
  • T-cells are isolated and transformed to carry a TAT-nuclease hybrid protein encoding gene.
  • the cells are amplified and replaced in the patients blood stream.
  • Such an approach may lead to a gene therapy for the treatment of AIDS: providing HIV-resistant T- cells.
  • RNA cleaving reagents combined with the above- described oligonucleotide cleaving agents may provide a two-pronged attack against viral diseases by providing cleavage of viral RNA and pro-viral DNA.
  • Synthetic oligonucleotide linkers and primers were prepared using commercially available auto- mated oligonucleotide synthesizers. Alternative ⁇ ly, custom designed synthetic oligonucleotides may be purchased, for example, from Synthetic Genetics (San Diego, CA) .
  • Oligonucleotide sequences encoding peptides can be either synthesized directly by standard methods of oligonucleotide synthesis, or, in the case of large coding sequences, synthesized by a series of cloning steps involving a tandem array of multiple oligonucleotide fragments correspond ⁇ ing to the coding sequence (Crea; Yoshio et al.; Eaton et al.). Oligonucleotide coding sequences can be expressed by standard recombinant proce ⁇ dures (Sambrook et al.; Ausubel et al.)
  • peptides can be synthesized directly by standard in vitro techniques (Applied Biosystems, Foster City CA) .
  • T7 RNA polymerase was purchased from Promega (Madison, WI) and used as per the manufacturer's instructions.
  • Polynucleotide kinase and restriction enzymes were obtained from Boehringer Mannheim (Indianapolis IN) or New England Biolabs (Beverly MA) and were used as per the manufacturer's directions.
  • Ribonuclease Tl, Ribonuclease CL3, and SI nuclease were obtained from Boehringer Mannheim and were used as per the manufacturer's direc ⁇ tions.
  • Uranyl nitrate was obtained from Mallencrodt (Paris KY) .
  • Radionuclides were obtained from New England Nuclear (Boston, Mass.), ICN (Costa Mesa CA) or Amersham (Arlington Heights IL) .
  • TAT and REV proteins are isolated as previously described (Weeks et al., 1990, herein incorporated by reference; Brown et al., 1990, herein incorporated by reference) .
  • the entire protein coding sequences of the TAT and REV proteins are presented in Figures 2A (SEQ ID N0:1) and 4 (SEQ ID NO:6), respectively, polypeptides derived from these proteins were synthesized at SRI's polypeptide synthesis facility using a Beck an model 990 polypeptide synthesizer, as per the manufacturer's instructions.
  • Polypeptides having the same C-terminal sequence but truncated at different N-terminal sites are recovered from a single solid-phase synthesis by removing some of the reaction bed at different stages of the syn- thesis.
  • cysteine residue can be added at any position internal to the polypeptide sequence or at either the amino- or carboxy- terminal ends of the protein.
  • the insertion of cysteine groups must be consistent with the maintenance of RNA-binding activity; such binding properties can be tested as described below.
  • TAT24C The TAT polypeptide designated TAT24C was chemically synthesized as just described.
  • TAT24C consists of amino acid residues 49-72 of the TAT protein (Figure 2A, underlined sequence) and an additional cysteine residue at the C-terminus ( Figure 2B, SEQ ID NO:2).
  • the TAT24C polypeptide was purified by standard HPLC.
  • the resulting polypeptides were separated from un-reacted iodo compound by passing the reaction mixtures through Sephadex G-50 spin columns (Pharmacia, Piscataway NJ) .
  • HIV-1 TAR is the 57-nt RNA stem-loop structure found in HIV-1 mRNA (nt 1-57); HIV-2 TAR includes the region of HIV-2 RNA essential for transactivation by HIV-2 TAT (nt 13-91, Arya et al., 1988); and ⁇ TAR is a truncated RNA containing the minimum TAT binding site (nt 17-43, Weeks et al., 1990 ).
  • the three RNA substrates are shown in Figures 1A, IB, and lC.
  • RNA substrates are presented in the sequence listing as follows: HIV-1 TAR, SEQ ID NO:3; HIV-2 TAR, SEQ ID NO:4; and ⁇ TAR, SEQ ID NO:5.
  • the RNA substrates were synthesized as follows. Synthetic DNA templates were formed by standard phosphoramidate synthesis using a Model 381B synthesizer (Applied Biosystems, Foster City, CA) . The synthetic DNA templates, containing T7 promoter sequences (Stahl et al. , 1981; Davanloo et al.
  • RNA molecules were purified by size fractionation on denaturing 10% polyacrylamide gels (Maniatis et al. ; Sambrook et al.) followed by electroelution. The isolated RNA molecules were then heated to 70'C and slowly cooled to room temperature to facilitate formation of the native secondary structure.
  • WT-RRE wild-type RRE
  • ⁇ RRE truncated version of RRE
  • ⁇ RRE truncated version of RRE
  • RNAs are synthesized by in vitro transcription using T7 RNA polymerase as described above for TAR RNAs.
  • WT-RRE can also be transcribed using a "BLUESCRIPT" plasmid (Stratagene, La Jolla, CA) carrying a 280 bp insert containing base pairs 7333-7612 of the RRE region (Daly et al. , 1989) . Transcribed RNAs are purified and end-labeled as described above.
  • RNA substrate Approximately 10 3 cpm of each uniformly labelled RNA substrate (Example 2) was incubated with either TAT24C or TAT24C-phen in a buffer containing 70 mM NaCl, 0.2 mM EDTA, 10 mM Tris-HCl (pH 7.5), 5% glycerol and 0.1% "NONIDET P40" (Sigma, St. Louis MO) for 20 minutes at 25°C. The samples were then run on a 10% native polyacrylamide gels in TBE (Tris-Borate-EDTA) buffer (Maniatis et al.; Sambrook, et al.) at room temperature. To obtain autoradiograms the gels were exposed to X-ray film. Figure 3 shows a photograph of the resulting autoradiogram.
  • TBE Tris-Borate-EDTA
  • panel A using HIV-1 TAR-RNA substrate: lane 1, in the presence of TAT24C (200 ng) ; lane 2, no polypeptide was added; lane 3, in the presence of TAT24C-phen (200 ng) .
  • panel B using HIV-2 TAR-RNA substrate: lane 1, in the presence of TAT24C (200 ng) ; lane 2, no polypeptide was added; lane 3, in the presence of TAT24C-phen (200 ng) .
  • panel C using ⁇ TAR-RNA as substrate: lane 1, no polypeptide was added; lane 2, in the presence of TAT24C-phen (200 ng) .
  • panel D using yeast tRNA (Bethesda Research Laboratories, Gaithersburg MD) as the substrate: lane 1, in the presence of TAT24C-phen (200 ng) ; lane 2, no protein was added; lanes 3 and 4 were with 200 ng and 400 ng of TAT24, respectively.
  • yeast tRNA Bethesda Research Laboratories, Gaithersburg MD
  • Figure 3C lane 2
  • These results show binding of polypeptides to all three RNAs containing the TAT responsive TAR element.
  • the mobility shift of samples containing modified and unmodified polypeptides are virtually identical, indicating that the attachment of the phenanthroline moiety at the C-terminus of the polypeptide does not affect binding to the TAR site.
  • Example 4 Cleavage of TAR-Site Containing Substrates
  • Cleavage of HIV-1 TAR Cleavage reactions using the polypeptide- cleaving reagents of the present invention were typically performed as follows. RNA substrates were 5' end-labeled (Maniatis et al. ; Sambrook, et al.) employing T4 polynucleotide kinase (Boehringer Mannheim) using 32 P ⁇ 7-ATP (Amersham) and purified by gel electrophoresis as described above.
  • RNA was incubated in 10 ⁇ l of buffer A in the presence of TAT24C-phen, and cleavage was initiated by adding CuS0 4 (to a final concentration of 10 ⁇ M) and mercaptopropionic acid (to a final concentration of 2.5 mM) after 10 minutes at 22 * C. After incubating for 17 hours, the reaction was stopped by adding the following to the indicated final concentrations: 2,9-dimethyl-l,10-phenanthroline to 3 mM, tRNA to 0.2 mg/ml, and sodium acetate (NaOAc) to 0.3 M.
  • NaOAc sodium acetate
  • TAT24C-phen (20 pmol); lane 3, TAT24C-phen (30 pmol) ; lane 4, SI nuclease (1 U) , incubated for 5 min at room temperature; lane 5, G-specific reaction, ribonuclease Tl (1 U) , incubated for 10 min at 37"C in 10 ⁇ l of buffer A (70 mM NaCl, 10 mM Tris-HCl (pH 7.5)); lane 6, C-specific reac ⁇ tion, ribonuclease CL3 (0.2 U) , incubated for 20 min at 37°C in 10 ⁇ l of buffer A; lane 7, cleavage at every nucleotide by irradiating RNA with 350-nm light (1.2 J in a "STRATALINKER", Stratagene, La Jolla, CA) at 25°C in the presence of 20 mM uranyl nitrate; lane a, 40 ⁇ M Cu(II)-1,10
  • Figure 8B presents fine mapping analysis of the cleavage sites at the 3' half of HIV-1 TAR. All reactions were conducted as described above.
  • lane 1 incubation with TAT24C-phen (90 ng) ;
  • lane 2 incubation with TAT24C-phen (60 ng) ;
  • Figure 8B. represents a gel subjected to longer electrophoresis time in order to separate larger fragments.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed. The cleavage sites on the HIV-2 TAR RNA are indicated by arrows.
  • RNAs from all reactions were ethanol precipitated and analyzed on a denaturing (8.3 M urea) 15% polyacrylamide gel. The gel was dried and autoradiographed.
  • E. Analysis of Cleavage Results The chemical nuclease activity of Cu(II)-com ⁇ plexed 1,10-phenanthroline derives from an oxidative attack on the sugar ring by a copper-oxo species generated in the presence of a reducing agent (Sigman et al., 1990).
  • the nucleolytic activity of TAT24C-phen on HIV-1 TAR is shown in Figure 8A. As seen in lanes 2 and 3, cleavage occurs primarily in the loop of the target RNA (structure shown between Figures 8A and 8B) , especially at the uridine (U 30 ) in the 5' side of the loop.
  • a secondary cleavage site can also be seen on the stem, at nt 12-14 and 18 (indicated by the short arrows in the RNA structure shown in Figure 8A) and at nt 43-45 on the complementary region ( Figure 8B) .
  • the cleavage pattern on opposite sides of the stem is shifted to the 5' side, an indication that the cleaving moiety is occupying the major groove of the duplex RNA stem (Dervan,
  • the cleavage sites lie on either side of the bulge where the TAT protein is known to bind to the TAR target site ( Roy et al., 1990; Cordingley et al., 1990; Dingwall et al., 1989; Weeks et al., 1990). Because Cu(II)-phenanthroline is known to preferentially cleave unpaired bases of RNA (Murakawa et al., 1989), HIV-1 TAR was incubated with free Cu(II)-1,10-phenanthroline, i.e., not bound to RNA-binding protein, as a control.
  • TAT24C-phen produces no cleavage (lane B, Figure 8A) .
  • HIV-2 TAR In contrast to cleavage of HIV-1 TAR, for which the primary site is at the loop adjacent to the TAT binding site, cleavage of HIV-2 TAR takes place mainly at the stem, roughly midway between the two loops ( Figure 9A, lanes 1 and 2) .
  • HIV-2 TAR has two 2-nt bulges, both of which have the consensus TAT binding motif (Weeks et al. , 1990; Green et al., 1988; Frankel et al. , 1988; Milligan et al.,1987; Arya et al. , 1988; Weeks et al. , 1991; Murakawa et al., 1989).
  • HIV-1 TAT can transactivate HIV-2 LTR-directed gene expression when either stem-loop I or stem-loop II is present (although the TAT product of HIV-2 requires both stem-loops for efficient transactivation (Arya et al. , 1988; Emerman et al. , 1987). Accordingly, the HIV-1 encoded TAT protein appears to bind to either stem-loop. Because the TAT24C polypeptide is based on HIV-1 TAT, the cleavage of both loops probably results from binding of the polypeptide to both elements. The major cleavage site for HIV-2 thus corresponds to the minor cleavage site for HIV-1 TAR (i.e., approximately 3-8 base pairs from the bulge in the direction away from the loop(s)).
  • TAT24C-phen might bind only to stem-loop I and the molecule could fold to bring the two loops close together.
  • tRNA was used as the substrate for cleavage, the cleavage pattern induced by TAT24C-phen was identical with that caused by free Cu(II)-phenanthroline ( Figure 9B) , indicating that TAT24C-phen does not induce site-specific cleavage on RNA lacking a TAR site.
  • ADDRESSEE SRI International
  • MOLECULE TYPE RNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • (C) INDIVIDUAL ISOLATE a peptide derived from the REV protein of HIV-1
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • MOLECULE TYPE RNA (genomic)
  • CAGUGGGAAU AGGAGCUUUG UUCCUUGGGU UCUUGGGAGC AGCAGGAAGC ACUAUGGGCG 60
  • CAGCGUCAAU GACGCUGACG GUACAGGCCA GACAAUUAUU GUCUGGUAUA GUGCAGCAGC 120
  • MOLECULE TYPE RNA (genomic)

Abstract

Cette invention concerne la production d'agents de clivage de l'ARN dirigés sur un site. Ces agents sont des protéines de liaison de l'ARN ou des polypeptides dérivés de ces dernières, qui sont modifiés pour contenir une fraction capable de couper les squelettes de l'ARN. Dans une autre forme d'exécution, les agents sont des oligonucléotides à squelettes résistant aux nucléases sur lesquels on a fixé une fraction capable de couper les squelettes de l'ARN. Cette invention concerne également un procédé permettant de couper des substrats d'ARN cible à l'aide des agents de clivage décrits, ainsi qu'un procédé utilisé pour inhiber l'expression de l'ARN dans des cellules infectées.
PCT/US1992/010770 1991-12-13 1992-12-11 Reactifs antiviraux a base de proteines de liaison de l'arn WO1993012234A1 (fr)

Applications Claiming Priority (4)

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US07/808,452 US6063612A (en) 1991-12-13 1991-12-13 Antiviral reagents based on RNA-binding proteins
US07/808,452 1991-12-13
US82693492A 1992-01-21 1992-01-21
US07/826,934 1992-01-21

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US5599535A (en) * 1995-06-07 1997-02-04 Regents Of The University Of California Methods for the cyto-protection of the trabecular meshwork
WO2000037635A2 (fr) * 1998-12-22 2000-06-29 Subsidiary No. 3, Inc. Elements de suppresseur genetiques agissant contre le virus de l'immunodeficience humaine
US6171788B1 (en) 1997-01-28 2001-01-09 The Regents Of The University Of California Methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
JP2001199997A (ja) * 2000-01-21 2001-07-24 Kansai Tlo Kk 細胞透過性キャリアペプチド
US6465176B1 (en) 1998-10-02 2002-10-15 Message Pharmaceuticals, Inc. Method for identifying compounds RNA/RNA binding protein interactions
US6475724B1 (en) 1997-01-28 2002-11-05 The Regents Of The University Of California Nucleic acids, kits, and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US7138511B1 (en) 1997-01-28 2006-11-21 The Regents Of The University Of California Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US7696342B1 (en) * 2002-11-26 2010-04-13 Rosetta Genomics, Ltd. Bioinformatically detectable group of novel viral regulatory genes and uses thereof

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WO1996013608A2 (fr) * 1994-10-28 1996-05-09 Innogenetics N.V. Sequences d'acide polynucleique utilisees dans la detection et la differenciation d'organismes procaryotes
US6355450B1 (en) 1995-04-21 2002-03-12 Human Genome Sciences, Inc. Computer readable genomic sequence of Haemophilus influenzae Rd, fragments thereof, and uses thereof
US20030032610A1 (en) 1996-06-03 2003-02-13 Gilchrest Barbara A. Method to inhibit cell growth using oligonucleotides
FR2744134B1 (fr) 1996-01-29 1998-04-03 Biocem Proteines de reserve de plantes enrichies en acides amines, notamment alpha-zeine de mais enrichie en lysine plantes exprimant ces proteines
DE10051628B4 (de) * 2000-10-18 2007-06-06 Fresenius Hemocare Beteiligungs Gmbh Einzelsträngiges Oligonukleotid und dessen Verwendung
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599535A (en) * 1995-06-07 1997-02-04 Regents Of The University Of California Methods for the cyto-protection of the trabecular meshwork
US6171788B1 (en) 1997-01-28 2001-01-09 The Regents Of The University Of California Methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US6475724B1 (en) 1997-01-28 2002-11-05 The Regents Of The University Of California Nucleic acids, kits, and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US7138511B1 (en) 1997-01-28 2006-11-21 The Regents Of The University Of California Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US6465176B1 (en) 1998-10-02 2002-10-15 Message Pharmaceuticals, Inc. Method for identifying compounds RNA/RNA binding protein interactions
WO2000037635A2 (fr) * 1998-12-22 2000-06-29 Subsidiary No. 3, Inc. Elements de suppresseur genetiques agissant contre le virus de l'immunodeficience humaine
WO2000037635A3 (fr) * 1998-12-22 2001-10-18 Subsidiary No 3 Inc Elements de suppresseur genetiques agissant contre le virus de l'immunodeficience humaine
JP2001199997A (ja) * 2000-01-21 2001-07-24 Kansai Tlo Kk 細胞透過性キャリアペプチド
US7696342B1 (en) * 2002-11-26 2010-04-13 Rosetta Genomics, Ltd. Bioinformatically detectable group of novel viral regulatory genes and uses thereof

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