LAUSR

ConspectusRNA-based technologies to control gene expression, such as RNA interference (RNAi) and CRISPR-Cas9, have become powerful tools in molecular biology and genomics. The exciting potential that RNAi and CRISPR-Cas9 may also become new therapeutic approaches has reinvigorated interest in chemically modifying RNA to improve its properties for in vivo applications. Chemical modifications can improve enzymatic stability, in vivo delivery, cellular uptake, and sequence specificity as well as minimize off-target activity of short interfering RNAs (siRNAs) and CRISPR associated RNAs. While numerous good solutions for improving stability toward enzymatic degradation have emerged, optimization of the latter functional properties remains challenging. In this Account, we discuss synthesis, structure, and biological activity of novel nonionic analogues of RNA that have the phosphodiester backbone replaced by amide linkages (AM1). Our long-term goal is to use the amide backbone to improve the stability and specificity of siRNAs and other functional RNAs. Our work in this area was motivated by early discoveries that nonionic backbone modifications, including AM1, did not disturb the overall structure or thermal stability of RNA duplexes. We hypothesized that the reduced negative charge and hydrophobic nature of the AM1 backbone modification might be useful in optimizing functional applications through enhanced cellular uptake, and might suppress unwanted off-target effects of siRNAs. NMR and X-ray crystallography studies showed that AM1 was an excellent mimic of phosphodiester linkages in RNA. The local conformational changes caused by the amide linkages were easily accommodated by small adjustments in RNA's conformation. Further, the amide carbonyl group assumed an orientation that is similar to one of the nonbridging P-O bonds, which may enable amide/phosphate mimicry by conserving hydrogen bonding interactions. The crystal structure of a short amide-modified DNA-RNA hybrid in complex with RNase H indicated that the amide N-H could also act as an H-bond donor to stabilize RNA-protein interactions, which is an interaction mode not available to phosphate groups. Functional assays established that amides were well tolerated at internal positions in both strands of siRNAs. Surprisingly, amide modifications in the middle of the guide strand and at the 5'-end of the passenger strand increased RNAi activity compared to unmodified siRNA. Most importantly, an amide linkage between the first and second nucleosides of the passenger strand completely abolished its undesired off-target activity while enhancing the desired RNAi activity. These results suggest that RNAi may tolerate more substantial modifications of siRNAs than the chemistries tried so far. The findings are also important and timely because they demonstrate that amide modifications may reduce off-target activity of siRNAs, which remains an important roadblock for clinical use of RNAi. Taken together, our work suggests that amide linkages have underappreciated potential to optimize the biological and pharmacological properties of RNA. Expanded use of amide linkages in RNA to enhance CRISPR and other technologies requiring chemically stable, functional mimics of noncoding RNAs is expected.