Amide Modifications in the Seed Region of the Guide Strand Improve the On-Target Specificity of Short Interfering RNA.

RNA interference (RNAi) is a well-established research tool and is also maturing as a novel therapeutic approach. For the latter, microRNA-like off-target activity of short interfering RNAs (siRNAs) remains as one of the main problems limiting RNAi drug development. In this communication, we report that replacement of a single internucleoside phosphodiester in the seed region (nucleotides 2 to 7) of the guide strand with an amide linkage suppressed the undesired microRNA-like off-target activity by at least an order of magnitude. For the specific siRNA targeting the PIK3CB gene, an amide modification between the third and fourth nucleotides of the guide strand showed the strongest enhancement of specificity (completely eliminated off-target silencing) while maintaining high on-target activity. These results are important because off-target activity is one of the main remaining roadblocks for RNA based drug development.

Amide Internucleoside Linkages Are Well Tolerated in Protospacer Adjacent Motif-Distal Region of CRISPR RNAs.

The development of CRISPR-Cas9 mediated gene editing technology is revolutionizing molecular biology, biotechnology, and medicine. However, as with other nucleic acid technologies, CRISPR would greatly benefit from chemical modifications that optimize delivery, activity, and specificity of gene editing. Amide modifications at certain positions of short interfering RNAs have been previously shown to improve their RNAi activity and specificity, which motivated the current study on replacement of selected internucleoside phosphates of CRISPR RNAs with amide linkages. Herein, we show that amide modifications did not interfere with CRISPR-Cas9 activity when placed in the protospacer adjacent motif (PAM) distal region of CRISPR RNAs. In contrast, modification of the seed region led to a loss of DNA cleavage activity at most but not all positions. These results are encouraging for future studies on amides as backbone modifications in CRISPR RNAs.

A synthetic dolastatin 10 analogue suppresses microtubule dynamics, inhibits cell proliferation, and induces apoptotic cell death.

We have synthesized eight analogues (D1-D8) of dolastatin 10 containing several unique amino acid subunits. Of these agents, D5 was found to be most effective in inhibiting both HeLa cell proliferation and microtubule assembly in vitro. At low nanomolar concentrations, D5 inhibited the proliferation of several types of cancer cells in culture. D5 bound to tubulin with a dissociation constant of 29.4 ± 6 µM. D5 depolymerized microtubules in cultured cells and produced mulitpolar spindles. At its half-maximal inhibitory concentration (15 nM), D5 strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. D5 increased the accumulation of checkpoint proteins BubR1 and Mad2 at the kinetochoric region and caused G2/M block in these cells. The blocked cells underwent apoptosis with the activation of Jun N-terminal kinase. The results suggested that D5 exerts its antiproliferative action by dampening microtubule dynamics.

Total synthesis of (29S,37S)-isomer of malevamide E, a potent ion-channel inhibitor.

The first total synthesis of (29S,37S)-malevamide E (1), a potent ion channel inhibitor, has been achieved in a convergent fashion involving Julia-Kocienski olefination, Urpi acetal aldol and Shiina macrolactonization reactions as the key steps. The strategy developed herein is amenable for the synthesis of the other possible isomers in search for the correct stereoisomer of the naturally occurring molecule.

Studies directed toward the development of amide-linked RNA mimics: synthesis of the monomeric building blocks.

A general approach toward the synthesis of all four monomeric building blocks of the ribonucleoside amino acids 3'-amino-5'-carboxymethyl-3',5'-dideoxy nucleosides in their protected forms is described that will facilitate the development of amide-linked RNA mimics.