Efficient synthesis and cell-based silencing activity of siRNAS that contain triazole backbone linkages

Synthesis and in vitro assessment of chemically modified siRNAs targeting BCL2 that contain 2′-ribose and triazole-linked backbone modifications

“…7 SiRNAs are also prone to cause off-target effects, leading to suppression of non-target mRNA sequences. [15][16][17] When this backbone modification was incorporated into an siRNA, gene-silencing data illustrated dose-dependent knockdown of an exogenous gene in vitro and improved serum nuclease resistance when compared to an unmodified siRNA. These limitations are often due to the native structure of RNA which is recognized by endogenous proteins.…”

“…8 Finally, siRNAs have difficulty being internalized by cells, thus making their bioavailability a challenge. 16 Oligonucleotide therapeutics which are commercially available contain multiple types of chemical modifications within their structure, but currently no universally acceptable chemical modification design exists for siRNAs, despite some significant advances in the field. However, through the use of chemical modifications, many of these pharmacological shortcomings and limitations can be modulated.…”

“…We have previously described optimal placement of U t U modifications within siRNAs, 16 thus U t U modifications were placed at either the 3′-overhang position or within the 3′region of the sense strand. The 2′-ribose modifications were placed in siRNA duplexes either with or without U t U modifications.…”

“…11,16 Unmodified and modified siRNAs were incubated in fetal bovine serum (FBS) for up to five hours to analyze the truncated products in a time-dependent manner using polyacrylamide gel electrophoresis (Fig. Independently, it has been previously reported that both U t U and 2′-ribose modifications incur nuclease stability when compared to unmodified siRNAs.…”

“…6,11,16 Nuclease stability was additive when combining triazole-linked backbone modifications with 2′-ribose modifications, especially when the triazole modification was placed at the overhang position. The qPCR BCL2 screens show that these modifications have enhanced gene-silencing activity either independently or in conjunction within the same siRNA.…”