Muthiah Manoharan

Previously, we used an aminooxy click chemistry (AOCC) strategy to introduce pairs of ligands into oligonucleotides during solid-phase synthesis by using nucleoside and non-nucleoside building blocks. However, the standard oligonucleotide synthesis conditions were incompatible with the incorporation of certain complex ligands. In this report, first, a post-oligonucleotide synthesis solution-phase AOCC approach was developed to address this issue. Different pairs of ligands were conjugated to oligonucleotides through an O-amino building block incorporated during the solid-phase synthesis. Second, a small interfering RNA with a sense strand conjugated using the AOCC strategy to target the ApoB gene using a receptor-targeting ligand (GalNAc) and a pharmacokinetic enhancer (C16 alkyl) was evaluated in mice. The bis-heteroconjugate had a longer duration of action and higher level of silencing than the siRNA carrying only the receptor-targeting ligand.

α-(l)-(3′–2′)-Threofuranosyl nucleic acid (TNA) cross-pairs with DNA and RNA and is not degraded by nucleases and thus has potential for use in diagnostics and therapeutics based on nucleic acids. Here, we describe the synthesis of 15 building blocks of chiral 4′-(R)- and 15 building blocks of chiral (S)-methyl-α-(l)-threofuranosyl nucleic acid (4′-Me-TNA) monomers and their incorporation into oligonucleotides. Crystallographic studies of RNAs containing 4′-Me-TNA modifications revealed the impact of the methyl substituent on the sugar pucker and duplex conformation. Biostability studies showed that a terminal 4′-Me-TNA modification on a deoxyoligonucleotide imparted resistance to nuclease digestion equivalent to unmethylated TNA, which is markedly superior to that of more conventional 2′-ribo and 2′-deoxy modifications. A single nucleotide incorporation of the 4′-(R)-Me-TNA destabilized an otherwise all-RNA 12-mer duplex by 6.5 to 12 °C, depending on the base, relative to the unmodified RNA control duplex; the (S) isomer was less destabilizing than the (R) isomer. Computational models were used to rationalize the high levels of nuclease resistance and the thermal destabilization. The 4′-Me-TNA triphosphates are poorly recognized by an engineered TNA polymerase making it unlikely that the 4′-Me-TNA triphosphates will be recognized by natural polymerases. Together, these properties warrant the evaluation of 4′-Me-TNA in nucleic acid-based therapeutics.

We describe a strategy for the synthesis of DNA, RNA and 2′-O-methyl (2′-O-Me) oligonucleotides containing 2-fluoro-adenine (AF) and isoguanine (iG) nucleobases. To the best of our knowledge, this is the first nonenzymatic route for the synthesis of oligonucleotides containing 2-fluoro-adenine. We also describe the hydrolytic strategy for the conversion of 2-fluoro-adenine into isoguanine. Unlike 2,6-diaminopurine, 2-fluoro-adenine and isoguanine modifications are thermally destabilizing in the context of DNA, RNA, and 2′-O-methyl oligonucleotide duplexes.

The C-5 position of pyrimidines is a known modification site for tuning the biophysical properties of oligonucleotides and nucleic acid duplexes. Consecutive C-5 phenyl triazole motifs in morpholino oligonucleotides boost duplex stability with RNA.

Similar in structure to both glycol nucleic acid (GNA) and peptide nucleic acid (PNA), serinol nucleic acid (SNA) combines the acyclic flexibility of GNA with the amide bond found in PNA and can form stable heteroduplexes with DNA and RNA. While the thermal and metabolic stability of SNA and SNA-modified heteroduplexes has been previously reported, we here describe the application of SNA in RNA interference (RNAi) using GalNAc-conjugated small interfering RNAs (siRNAs). Single incorporations were evaluated at each position across both the guide and passenger strands and subsequently evaluated at key positions in mice. Additionally, the off-target potential of siRNAs containing SNA was assessed. We demonstrate the position-dependent tolerance of SNA inside both the guide and passenger strands, and more specifically, high tolerance of SNA incorporation in the seed region of the guide strand, preserving on-target activity while mitigating microRNA-like off-target effects. Furthermore, the crystal structure of an RNA dodecamer containing a single SNA nucleotide was obtained and the resulting SNA structure was used to explain the activity of SNA containing siRNAs. Thus, SNA constitutes another viable modification in the siRNA toolbox for the development of potent and specific RNAi therapeutics.

5′-( E )- and 5′-( Z )-vinylphosphonate carbocyclic DNA and 5′-( E )-vinylphosphonate 2′- and 3′- O -methyl carbocyclic RNAs were incorporated at 5′ termini of antisense strands of small interfering RNAs.

Nucleotides with a carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and N1-methylpseudouridine; these modifications were reported to slow the degradation of large RNAs, but the mechanism is unknown. We measured kinetics of spontaneous and enzymatic cleavage at a single bond of synthetically modified RNAs and found that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and small alcohols of varied pK _a suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher pK _a and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies.

Inspired by the uncommon furanose configuration of 3'-deoxyapio-containing nucleic acids (apioNAs), we developed a facile and convenient synthesis of 24 building blocks of this modified nucleic acid monomer, including phosphoramidites, H-phosphonates, solid supports, and nucleoside triphosphates. The building blocks included those containing the four canonical RNA bases A, G, U, and C as well as T and 5-methyl-C and were synthesized starting from a single common sugar intermediate derived from d-(+)-xylose. DNA and RNA duplexes with a single apioNA modification in one strand were less thermodynamically stable than unmodified DNA or RNA. The crystal structure of apioNA-modified RNA octamer showed that the apioNA residue adopts an RNA-like structure but local reorientation of the apioNA sugar and 2'-phosphate and the difference in helical rise on the 5' side of the apioNA T relative to RNA likely contribute to the destabilizing effect of apioNA residues. At the terminus of a DNA strand, this modification provides extremely high resistance against both 3'- and 5'-exonucleases even when linked to the adjacent residue by a phosphodiester moiety. Molecular modeling of a DNA duplex containing apioNA was used to rationalize the DNA duplex destabilization and the exonuclease resistance resulting from incorporation of the apioNA residue. Use of apioNA NTPs as substrates for previously engineered α-l-threofuranosyl polymerases depends on both the enzyme and the nucleobase. These data indicate that apioNAs warrant further evaluation, and the building blocks synthesized will allow incorporation of apioNA into therapeutic oligonucleotides.

Unlocked nucleic acid (UNA) is a nucleic acid analog that has an acyclic ribose ring lacking the bond between C2′ and C3′ atoms. The base-pairing properties of UNA have been studied, and it has been used as a scaffold for conjugation, but the chemical space around UNA and its potential applications in the context of short interfering RNAs (siRNAs), which mediate RNA interference, have not been thoroughly explored. In this study, we report syntheses of methylated and methoxylated UNAs and their incorporation into siRNAs. siRNAs with 5′-(R)-methyl-UNA and with 5′-(S)-methyl-UNA in the seed region had comparable potencies but reduced off-target effects compared to siRNA modified with UNA. In mice, siRNAs with modified UNAs were of comparable potency to an siRNA of the same sequence and chemistry lacking UNA. Modeling studies indicated that the flexibilities of UNA and the modified UNAs facilitate kinking of the antisense strand when incorporated at position 7. These findings highlight the potential of modified UNA for advancing therapeutics that act through the RNA interference pathway.

Small interfering RNAs (siRNAs) are a proven therapeutic approach for controlling gene expression in the liver. Expanding the clinical potential of RNA interference requires developing strategies to enhance delivery to extra-hepatic tissues. In this study, we examine inhibiting transthyretin (TTR) gene expression by siRNAs in the eye. Anti-TTR siRNAs have been developed as successful drugs to treat TTR amyloidosis. When administered systemically, anti-TTR siRNAs alleviate symptoms by blocking TTR expression in the liver. However, TTR amyloidosis also affects the eye, suggesting a need for reducing ocular TTR gene expression. Here, we demonstrate that pyrimidine C5- and 2′-O-linked lipid-modified siRNAs formulated in saline can inhibit TTR expression in the eye when administered locally by intravitreal injection. Modeling suggests that length and accessibility of the lipid chains contribute to in vivo silencing. GalNAc-modified siRNAs also inhibit TTR expression, albeit less potently. These data support lipid-modified siRNAs as an approach to treating the ocular consequences of TTR amyloidosis. Inhibition of TTR expression throughout the eye demonstrates that lipid–siRNA conjugates have the potential to be a versatile platform for ocular drug discovery.