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To evaluate potential improvement in tissue specific targeting and cellular uptake of therapeutic ribozymes, we have developed three new phosphoramidite reagents. These reagents can be used in automated solid-phase synthesis to produce oligonucleotide conjugates containing N-acetyl-D-galactosamine (targeting hepatocytes) and folic acid (targeting tumor). N-Acetyl-D-galactosamine was attached through a linker to both 2'-amino-2'-deoxyuridine and D-threoninol scaffolds, and these conjugates were converted to phosphoramidite building blocks. Incorporation of a D-threoninol-based monomer into ribozymes provided multiply labeled ribozyme conjugates. Attachment of the fully protected pteroic acid to the D-threoninol-6-aminocaproyl-L-glutamic acid construct afforded the folic acid conjugate, which was converted into the phosphoramidite and incorporated onto the 5'-end of the ribozyme.

2'-Modified pyrimidine nucleoside 5'-triphosphates comprising amino, imidazole and carboxylate functionality attached to the 5-position of the base were synthesized. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.

The MS2 coat protein binds specifically to an RNA hairpin formed within the viral genome. By soaking different RNA fragments into crystals of MS2 coat protein capsids it is possible to determine the X-ray structure of the RNA-protein complexes formed. Here we present the structure to 2.85 A resolution of a complex between a chemically modified RNA hairpin variant and the MS2 coat protein. This RNA variant has a substitution at the -5 base position, which has been shown previously to be pyrimidine-specific and is a uracil in the wild-type RNA. The modified RNA hairpin contains a pyridin-4-one base (4one) at this position that lacks the exocyclic 2-oxygen eliminating the possibility of forming a hydrogen bond to asparagine A87 in the protein. The 4one complex structure shows an unprecedented major conformational change in the loop region of the RNA, whereas there is almost no change in the conformation of the protein.

Chemically modified nucleotide analogs have gained widespread popularity for probing structure-function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 A resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3'-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs.

A series of novel 2'-modified nucleoside 5'-triphosphates was synthesized. The amino, imidazole, and carboxylate functionalities were attached to the 5-position of pyrimidine base of these molecules through alkynyl and alkyl spacers, respectively. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.

Previously developed '5-ribo' nuclease stabilized hammerhead motif was further refined by systematic incorporation of 1-(beta-D-xylofuranosyl) adenine (xA) and 1-(beta-D-xylofuranosyl) guanine (xG) in the place of conserved ribopurine residues of the catalytic core. Modified ribozymes substituted with xA at positions A15.1 and A6 demonstrated catalytic activity close to the parent stabilized ribozyme. Analogous guanosine substitutions at positions G5, G8, and G12 substantially lowered catalytic rates.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

VP55, the vaccinia poly(A) polymerase catalytic subunit, interacts with oligonucleotide primers via two uridylate recognition sites (Deng, L., and Gershon, P. D. (1997) EMBO J. 16, 1103-1113). Here, we show that the cognate RNA sequence comprises a 5'-rU2-N15-rU-3' motif (where N = any deoxyribo or ribonucleotide), embedded within oligonucleotide primers 29-30 nucleotides (nt), or greater, in length. Nine residues separate the 3'-most ribouridylate of the optimally positioned motif from the primer 3'-OH. A ribose sugar at the extreme 3'-terminal nucleotide of the primer is absolutely required for VP55's adenylyltransferase activity, but not for stable VP55-RNA interaction. A ribose at position -3 markedly stimulates both adenylyltransferase activity and stable binding. The use of uridine analogs indicated (i) those functional groups of the uracil base which contribute to stable VP55-primer interaction, and (ii) that VP55's ability to discriminate uracil from cytosine stems largely from the requirement for a protonated N3 nitrogen within the pyrimidine ring. The rU2-N15-rU motif was identified within the uridylate-rich 3' end of a naturally occurring vaccinia mRNA. However, oligonucleotides whose only internal uridylates comprised the motif supported only a 3-5-nt processive burst of oligo(A) tail addition, as opposed to the approximately 30-35-nt burst observed with the naturally occurring 3' end.

The role of individual functional groups on cytidine 17 in the hammerhead ribozyme was assessed by introducing modified pyrimidines into two kinetically well-characterized hammerheads. As long as the pyrimide ring size was maintained, the modifications had no effect on substrate binding, suggesting that the C17-C3 hydrogen bond observed in the X-ray structure is energetically neutral. However, modification of the exocyclic amino group and the carbonyl of C17 reduced the cleavage rate significantly, indicating that these groups are important in stabilizing the transition-state structure. C17 modifications did not affect the ratio of the forward and reverse reaction rates. Thus, unlike that believed previously, C17 is another one of many hammerhead residues critical in maintaining its active structure.

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5'-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2'-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

A site-specific chemical modification strategy has been employed to elucidate structure-function relationships at the only phylogenetically nonconserved position within the core of the hammerhead ribozyme (N7). Four different base substitutions at position 7 resulted in increased catalytic rates. A pyridin-4-one base substitution increased the rate of the chemical step up to 12-fold. These results are the first examples of chemical modifications within a catalytic RNA that enhance the rate of the chemical step. Four base substitutions resulted in decreased catalytic rates. The results do not correlate with proposed hydrogen bond interactions (Pley et al., 1994; Scott et al., 1995). This study demonstrates the utility of using unnatural nucleotide analogs-rather than mutagenesis with the four standard nucleotides alone-to elucidate structure-function relationships of small RNAs.

Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2'-O-Me nucleotides, 1-2 other 2'-modified nucleotides at positions U4 and U7, and a 3'-3'-linked nucleotide "cap." Eight 2'-modified uridine residues were introduced at positions U4 and U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 substitutions at U4 and U7, or 2'-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only approximately 0.1 min. The addition of a 3'-3'-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to > 260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

To enhance the resistance of 2-5A (pppA2'p5'A2'p5'A) to degradation by exo- and endonucleases, a phosphorodithioate analog was synthesized using a solid-phase phosphite triester approach with N6-benzoyl-5'-O-dimethoxytrityl-3'-O-t-butyldimethylsilyladenosine 2'-[S-(beta-thiobenzoylethyl)-pyrrolidinophosphorothioamidit e]. 5'-Monophosphorylation was accomplished with 2-[2-(4,4'-dimethoxytrityloxy)-ethylsulfonyl]ethyl-(2-cyanoe thyl)-(N,N- diisopropyl)-phosphoramidite. The resulting product, p5'A2'(s2p)- 5'A2'(s2p)5'A, was approximately 10-fold less effective as an activator of purified human recombinant 2-5A-dependent RNase than was 2-5A itself. This loss of activation ability was related directly to the loss of binding ability of the phosphorodiothioate analog. As predicted, p5'A2'(s2p)5'A2' (s2p)5'A was stable to snake venom phosphodiesterase and the nucleolytic activities of both human lymphoblastoid CEM cell extracts and human serum, under conditions that led to facile degradation of parent 2-5A. This nuclease stability permitted the observation of the CEM cell extracts and human serum phosphatase activity which led to 5'-dephosphorylation of p5'A2'(s2p)5'A2'(s2p)5'A.

In order to obtain agents with therapeutic indices superior to those of AZT, FLT, or D4T, several analogues of anti-HIV-1 nucleosides were synthesized. These include 2',3'-dideoxy-2',3' -difluoro-5-methyluridine (13), its arabino analogue 19, arabino-5-methylcytosine analogue 21, 3'-deoxy-2',3'-didehydro-2' -fluorothymidine (25), 3'-azido-2',3'-dideoxy-2'-fluoro-5-methyluridine (29), 2'-azido-3'-fluoro-2',3'-dideoxy-5-methyluridine (31), and 2'3'-dideoxy-2' -fluoro-5-methyluridine (37). These new nucleosides were screened for their activity against HIV and feline TLV in vitro. None of the compounds showed significant activity. It is interesting to note that such a small modification in the sugar moiety of active anti-HIV nucleosides (i.e., displacement of hydrogen by fluorine) almost completely inactivate the agents.

1-(3-Azido-2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (6, F-AZT) and 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)cytosine (12, F-DDC) were synthesized from the potent antiherpes virus nucleosides 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (1, FMAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) in the hope that introduction of a 2-"up"-fluoro substituent might potentiate the anti-HIV activity of AZT and DDC. FMAU (1) was converted in three steps into 2,3'-anhydro-1-(2-fluoro-2-deoxy-5-O-trityl-beta-D-lyxofuranosyl)thymine (4), which when treated with NaN3 followed by detritylation afforded 6. F-DDC was prepared by two methods. Tritylation of FIAC followed by treatment of the product with thiocarbonyldimidazole afforded the 5'-O-trityl-3'-O-(imidazolyl)thiocarbonyl nucleoside 9. Upon radical reduction of 9 with Bu3SnH and AIBN, 5'-O-trityl-DDC 10 was obtained. Compound 10 was detritylated to give 12, which (when obtained by this procedure) resisted crystallization, but the diacetate 12' was obtained in crystalline form. Alternatively, FAC (14) was converted into N4,O5'-dibenzoyl derivative 15, which was treated with thiocarbonyldiimidazole. Reduction of 16 with Bu3SnH/AIBN followed by debenzoylation afforded 12, which was obtained in crystalline form. F-AZT did not exhibit any significant activity against the human immunodeficiency virus (HIV) in vitro. F-DDC, however, showed activity against HIV-1, but the therapeutic index is much inferior to that of AZT.

A new synthesis of 5-(monofluoromethyl)- and 5-(difluoromethyl)-2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil (F-FMAU and F2-FMAU) is reported. 3',5'-Di-O-(tert-butyldiphenyl)silylated thymidine or FMAU was photochemically brominated with NBS to the corresponding alpha-monobromide, which was hydrolyzed to the 5-hydroxymethyl derivative. Further oxidation of the latter with MnO2 afforded the 5-formyluracil nucleoside. Treatment of these nucleosides with DAST in CH2Cl2 gave the protected alpha-fluorinated nucleosides. Desiylation with TBAF afforded the desired free nucleosides. Also, 5-(trifluoromethyl)-2'-deoxy-2'-fluoro-beta-D-arabinofuranosyluracil (F3-FMAU) was synthesized by copper-catalyzed trifluoromethylation of 5-iodo-2'-fluoro-ara-U (FIAU). These new nucleosides were studied in comparison with the corresponding 2'-deoxy-erythro-pentofuranosyl derivatives, for their inhibitory activity against cellular thymidylate synthase (TS) and [3H]TdR incorporation into DNA, cytotoxicity against HL-60 cells, and antiviral activity against herpes simplex types 1 and 2 (HSV-1 and -2). F2-TDR and F3-TDR strongly inhibited TS and were also quite cytotoxic and antiherpetic, whereas FTDR was only active in the antiviral assay. In the 2'-fluoroarabino series, fluorine substitution at the alpha-methyl function did not alter significantly the antiherpetic activity. Although FMAU and F-FMAU did not inhibit TS to any significant extent, F2-FMAU and F3-FMAU were weakly inhibitory. The latter nucleosides did not inhibit [3H]TDR incorporation into DNA, while all the other alpha-fluorinated thymine nucleosides inhibited the incorporation of radioactivity of [3H]TDR into DNA to various extents. F2-FMAU and F3-FMAU were about 2 orders of magnitude less cytotoxic against HL-60 cells than were F2-TDR and F3-TDR. The results strongly suggest that in both the 2'-deoxy-2'-fluoroarabino and the 2'-deoxy-erythro-pentofurano series the cytotoxic action of the alpha,alpha-difluoro and alpha,alpha,alpha-trifluoro derivatives may involve the inhibition of TS. The synthesis of [2-14C]F2-FMAU, as an experimental imaging agent, is also described. Unfortunately, the highly selective uptake of the labeled compound within infected brain regions previously noted with [2-14C]FMAU was not detected with the derivative [2-14C]F2-FMAU.

Seven purine nucleosides containing the 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl moiety were synthesized and tested for their antitumor activity. Direct condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl bromide (1) with N6-benzoyladenine in CH2Cl2 followed by saponification of the product afforded the adenine nucleoside (I, 2'-F-ara-A). Deamination of I with NaNO2 in HOAc gave the hypoxanthine analogue (II, 2'-F-ara-H). The 6-thiopurine nucleoside (III, 2'-F-ara-6MP) was prepared by condensation of 1 with 6-chloropurine by the mercury procedure followed by thiourea treatment and saponification of the product. Methylation of III gave the 6-SCH3 analogue (IV). Raney Ni desulfurization of III afforded the unsubstituted purine nucleoside (V, 2'-F-ara-P). Condensation of 1 with 2-acetamido-6-chloropurine by the silyl procedure afforded the protected 2-acetamido-6-chloropurine nucleoside which served as the precursor for both the guanine and 6-thioguanine nucleosides (VI, 2'-F-ara-G and VII, 2'-F-ara-TG, respectively). Thus, alkaline hydrolysis of the precursor gave VI. Thiourea treatment prior to alkaline hydrolysis gave VII. The new nucleoside, 2'-F-ara-G (VI) is found to be selectively toxic to human T-cell leukemia CCRF-CEM.

In order to determine if modification of the 5'-position reduces or abolishes the antiviral activity of 2'-fluoro-5-iodo-ara-C (FIAC), 2'-fluoro-5-iodo-ara-U (FIAU), or 2'-fluoro-5-methyl-ara-U (FMAU) against human cytomegalovirus (HCMV) and herpes simplex virus (HSV), the 5'-deoxy, 5'-mercapto, and 5'-amino analogues of these nucleosides were prepared. 5'-Deoxy-FIAC and 5'-deoxy-FIAU were prepared by catalytic hydrogenation of 5'-iodo-FIAC and 5'-iodo-FIAU to 5'-deoxy-FAC and 5'-deoxy-FAU, respectively, followed by reiodination at C-5. Reduction of 5'-iodo-FMAU afforded 5'-deoxy-FMAU. These 5'-deoxy nucleosides were found to be inactive against HCMV, indicating that the conversion to 5'-phosphate by the cellular enzyme(s) is a requirement for antiviral activity against this virus. Other 5'-modified (NH2 and SH) analogues were also prepared from 5'-O-tosyl-FIAC and 5'-O-tosyl-FMAU. Treatment of these tosylates with LiN3 in DMF afforded the corresponding 5'-N3 products. Catalytic hydrogenation of 5'-N3-FMAU afforded 5'-NH2-FMAU, whereas 5'-NH2-FIAC was obtained by treatment of 5'-N3-FIAC with Ph3P in pyridine. 5'-Mercapto analogues were prepared by treatment of 5'-O-tosyl-3'-O-acetyl nucleosides with KSAc followed by deacetylation. 5'-NH2-FMAU was the only compound that showed good activity against HSV-1 and HSV-2 in vitro. However, this compound was less potent and had a lower therapeutic index than FMAU.