We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.
We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.
An established paradigm in pre-mRNA splicing is the recognition of the 59 splice site (59ss) by canonical basepairing to the 59 end of U1 small nuclear RNA (snRNA). We recently reported that a small subset of 59ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, we now demonstrate that many other 59ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 59ss or U1 RNA strand, which we term ''bulge registers.'' By combining experimental evidence with transcriptome-wide free-energy calculations of 59ss/U1 base-pairing, we estimate that 10,248 59ss (~5% of human 59ss) in 6577 genes use bulge registers. Several of these 59ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing.[Keywords: 59 splice site; U1 small nuclear RNA; base-pairing register; bulged nucleotide; splicing mutation; alternative splicing] Supplemental material is available for this article. Pre-mRNA splicing is an essential processing step for the expression of ;90% of protein-coding human genes and relies on conserved sequence elements at both ends of introns, termed splice sites (Sheth et al. 2006;Wahl et al. 2009). These elements are highly diverse, considering that thousands of different sequences act as naturally occurring splice sites in the human transcriptome (Sahashi et al. 2007;Roca and Krainer 2009). The characterization of these sequence elements and the factors that recognize them has been essential for predicting exons in new genes, for the study of alternative splicing (Nilsen and Graveley 2010), and for classifying mutations in these elements that cause human genetic diseases (Buratti et al. 2007).Typically, the strength of a splice site (or splice site score) is estimated by algorithms that measure its concordance to matrices built using large collections of splice sites (Senapathy et al. 1990;Brunak et al. 1991;Yeo and Burge 2004;Sahashi et al. 2007;Hartmann et al. 2008). These methods implicitly assume that all of the sequences used to build the matrix are recognized by the same mechanism. However, there are cases in which the splice site score does not reflect the strength of the splice site determined experimentally (Roca and Krainer 2009), highlighting the limitations of these tools. Furthermore, the recognition mechanisms for many splice sites predicted to be weak are poorly understood.Splicing of >99% of pre-mRNA introns is catalyzed by the major spliceosome, a dynamic macromolecular machine composed of five small nuclear RNAs (snRNAs) and associated polypeptides, plus many other protein factors (Wahl et al. 2009). The U1 small nuclear ribonucleoprotein particle (snRNP), comprising the U1 snRNA and 10 polypeptides (Pomeranz K...
The synthesis, biophysical, structural and biological properties of both isomers of 3′-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with 3′-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer, provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an inter-nucleotide C-F…H-C pseudo hydrogen bond between F3′ of Ara-FHNA and C8-H of the nucleobase from the 3′-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)…H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.
We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in Tm, cell culture and animal experiments and compare them to their gap-matched 20-mer 2′-O-methoxyethyl (MOE) and 14-mer 2′,4′-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar Tm and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED50 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower Tm and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.
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