Amide-Modified RNA: Using Protein Backbone to Modulate Function of Short Interfering RNAs

Abstract

RNA-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 towards enzymatic degradation have emerged, optimization of the latter functional properties remains challenging. In this Account, we discuss synthesis, structure, and biological activity of novel non-ionic 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 non-ionic 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 non-bridging 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

miRNAs in Cancer (Review of Literature)

“…Secondly, the in vivo delivery of artificial miRNAs is a challenge, and no single, effective method of transporting them to the target tissue (specific route of delivery) has yet been proposed [ 174 ]. Thirdly, the use of synthetic miRNA carriers raises doubts, due to the possibility of their degradation in the blood and potential toxicity [ 175 , 176 , 177 , 178 ] and the durability of the effects of therapy.…”

Beyond ribose and phosphate: Selected nucleic acid modifications for structure–function investigations and therapeutic applications

“…The chemical make-up of RNA, i.e., the ribose-phosphate backbone, has inspired countless strategies to chemically modify either the sugar [ 12 , 41 – 44 ], or the phosphate (e.g., amide-RNA [ 45 ]), or both [ 46 – 47 ]. In addition, the ribose has been replaced with alternative sugar moieties, such as a tetrose (ʟ-α-threofuranose, TNA [ 48 ]), and hexoses (e.g., hexitol, HNA [ 49 ]; altritol AtNA [ 50 ]; xylol XyNA [ 51 ]), or cyclohexene (CeNA [ 52 ]), a morpholino moiety (PMO [ 53 ]), and an acyclic, chiral glycol linker (GNA [ 54 ]), to generate so-called xeno nucleic acids (XNAs [ 55 – 56 ]).…”

Hemocyte siRNA uptake is increased by 5′ cholesterol-TEG addition in Biomphalaria glabrata, snail vector of schistosome

“…The use of dsRNA remains a molecular tool of choice in functional genomics, especially when the CRISPR technology is not yet established for the studied organism. Several modifications can be grafted on short nucleic acid such as 2′ ribose substitutions ( Alagia & Eritja, 2016 ; Allerson et al, 2005 ; Malek-Adamian et al, 2019 ) or chemically-modified internucleotide phosphodiester ( Alagia & Eritja, 2016 ; Deleavey & Damha, 2012 ; Kotikam & Rozners, 2020 ) to increase gene-silencing efficiency. In addition, achieving high RNAi efficiency remains a challenge in particular to target genes in different tissues.…”