Chirality Dependent Potency Enhancement and Structural Impact of Glycol Nucleic Acid Modification on siRNA.

Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. In addition, the GNA-T nucleobase adopts a rotated conformation in which the 5-methyl group points into the minor groove, rather than the major groove as in a normal Watson-Crick base pair. This observation of reverse Watson-Crick base pairing is further supported by thermal melting analysis of GNA-C and GNA-G containing duplexes where it was demonstrated that a higher thermal stability was associated with isoguanine and isocytosine base pairing, respectively, over the canonical nucleobases. Furthermore, it was also shown that GNA nucleotide or dinucleotide incorporation increases resistance against snake venom phosphodiesterase. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc conjugate duplex targeting mouse transthyretin (TTR) indicated that GNA is well tolerated in the seed region of both strands in vitro, resulting in an approximate 2-fold improvement in potency. Finally, these conjugate duplexes modified with GNA were capable of maintaining in vivo potency when subcutaneously injected into mice.

Allele-selective inhibition of mutant atrophin-1 expression by duplex and single-stranded RNAs.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (ATN-1) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant ATN-1 were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (ATXN-3) and huntingtin (HTT). These data demonstrate that the expanded trinucleotide repeat within ATN-1 mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes.

Regiospecific solid-phase synthesis of branched oligoribonucleotides that mimic intronic lariat RNA intermediates.

We have developed new solid phase methods for the synthesis of branched RNAs that mimic intronic lariat RNA intermediates. These methods produce branched oligoribonucleotide sequences of arbitrary length, base composition, and regiochemistry at the branchpoint junction. The methods utilize branching monomers that allow for the growth of each branch regioselectively from any of the hydroxyl positions (5', 3', or 2') at the branch-point junction. The integrity and branchpoint connectivity of the synthetic products have been confirmed by HPLC and MS analysis, and cleavage of the 2',5' linkage by recombinant debranching enzyme. Nonhydrolyzable branched RNA analogues containing arabinose instead of ribose at the branchpoint junction were shown to inhibit debranching activity and, hence, represent "decoys" for sequestering RNA binding proteins thought to drive amyotrophic lateral sclerosis (ALS).

RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression.

Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing.

Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.

Multivalent cyclic RGD conjugates for targeted delivery of small interfering RNA.

We have designed, synthesized, and tested conjugates of chemically modified luciferase siRNA (Luc-siRNA) with bi-, tri-, and tetravalent cyclic(arginine-glycine-aspartic) (cRGD) peptides that selectively bind to the αvß3 integrin. The cellular uptake, subcellular distribution, and pharmacological effects of the cRGD-conjugated Luc-siRNAs compared to those of unconjugated controls were examined using a luciferase reporter cassette stably transfected into αvß3 positive M21(+) human melanoma cells. The M21(+) cells exhibited receptor-mediated uptake of cRGD-siRNA conjugates but not of unconjugated control siRNA. The fluorophore-tagged cRGD-siRNA conjugates were taken up by a caveolar endocytotic route and primarily accumulated in cytosolic vesicles. The bi-, tri-, and tetravalent cRGD conjugates were taken up by M21(+) cells to approximately the same degree. However, there were notable differences in their pharmacological effectiveness. The tri- and tetravalent versions produced progressive, dose-dependent reductions in the level of luciferase expression, while the bivalent version had little effect. The basis for this divergence of uptake and effect is currently unclear. Nonetheless, the high selectivity and substantial "knock down" effects of the multivalent cRGD-siRNA conjugates suggest that this targeting and delivery strategy deserves further exploration.

New light labile linker for solid phase synthesis of 2'-O-acetalester oligonucleotides and applications to siRNA prodrug development.

We report on the synthesis and properties of oligonucleotides containing 2'-O-(levulinic acid) and 2'-O-(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we believe that these derivatives will have potential use as nucleic acid prodrugs. In addition, we report on the synthesis of a novel solid support with a photolabile linker that not only allows for the synthesis of oligonucleotides containing various 2'-O-acetalesters, but can be generally adopted to the synthesis of base-sensitive oligoribonucleotides. The release of oligonucleotides from this support is faster than with conventional linkers.

Acetal levulinyl ester (ALE) groups for 2'-hydroxyl protection of ribonucleosides in the synthesis of oligoribonucleotides on glass and microarrays.

We describe a synthetic strategy that permits both the growth and deprotection of RNA chains that remain attached to a solid polymer support or chip surface. The key synthons for RNA synthesis are novel 5'-O-DMTr 2'-acetal levulinyl ester (2'-O-ALE) ribonucleoside 3'-phosphoramidite derivatives. In the presence of 4,5-dicyanoimidazole (DCI) as the activator, these monomers coupled to Q-CPG solid support with excellent coupling efficiency (approximately 98.7%). The method was extended to the light directed synthesis of poly rU and poly rA on a microarray through the use of a 5'-O-(2-(2-nitrophenyl)propoxycarbonyl)-2'-O-ALE-3'-phosphoramidite derivative. A two-stage deprotection strategy was employed to fully deblock the RNA directly on the Q-CPG or microarray support without releasing it from the support's surface: phosphate group deblocking with NEt(3) in acetonitrile (ACN) (2:3 v/v; 1 h, r.t.) followed by removal of the 2'-O-ALE groups under mild hydrazinolysis conditions (0.5-4 h, r.t.). This last treatment also removed the levulinyl (Lv) group on adenine (N(6)) and cytosine (N(4)) and the dimethylformamidine (dmf) group on guanine (N(2)). The chemistry and methods described here pave the way to the fabrication of microarrays of immobilized RNA probes for analyzing molecular interactions of biological interest.

The acetal levulinyl ester (ALE) group for the 2'-hydroxyl protection of ribonucleosides and the synthesis of oligoribonucleotides.

A novel 2'-O-ALE (Acetal Levulinyl Ester) uridine phosphoramidite derivative has been efficiently prepared and used for the solid-phase synthesis of a chimeric oligonucleotide strand, 5'-rU11-dT-3'. The average coupling yield of the phosphoramidite was comparable to one obtained with a 2'-TBDMS rU phosphoramidite reagent. Upon completion of the RNA chain assembly, the cyanoethyl phosphate protecting groups were removed using a solution of triethylamine in acetonitrile (2:3 v/v). The 2'-O-ALE protecting groups were cleaved under hydrazinolysis conditions. Finally, the RNA oligonucleotide was released from the Q-CPG support when treated with 1 molar TBAF in THF. When the TBAF step was carried out prior to hydrazinolysis, an oligonucleotide with its intact 2'-O-ALE groups was obtained.

Toward the discovery of new antifungal agents: the design and validation of a novel 2'P-RNA probe and high throughput screening assay against 2'-phosphotransferase Tpt1p.

We report the solid-phase synthesis of novel 2'P-RNA probes for use in fluorescence polarization (FP) ligand binding assays that screens for inhibitors of the yeast 2'- phosphotransferase Tpt1p. The probe was synthesized by utilizing silyl phosphoramidite chemistry and a phosphoramidite synthon containing an orthogonal (DMT) protecting group at its 2'-position. Regioselective removal of the 2'-DMT group and phosphitylation of the unmasked 2'-hydroxyl group afforded the desired 2'P-RNA sequence.

An intact unfolded protein response in Trpt1 knockout mice reveals phylogenic divergence in pathways for RNA ligation.

Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2'-PO4(2-) at the splice junction, which is subsequently removed by an essential 2'-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNA(Tyr) is fully functional in the Trpt1-/- mouse cells. These observations argue against the presence of a 2'-PO4(2-) at the splice junction of ligated RNA molecules in Trpt1-/- cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals.

Solid-phase synthesis and on-column deprotection of RNA from 2'- (and 3'-) O-levulinated (Lv) ribonucleoside monomers.

[reaction: see text] The solid-phase synthesis of oligoribonucleotides derived from ribonucleosides esterified at the 2'- (or 3'-) position with the levulinyl (Lv) group is described. The oligomers can be released from the solid support as 2'-O-Lv ester derivatives or fully deprotected while still attached to the solid support.