Effect of 2′-5′/3′-5′ phosphodiester linkage heterogeneity on RNA interference

Abstract

We report on the synthesis of siRNAs containing both 2′-5′- and 3′-5′-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2′-5′ linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5′-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5′p-rN1-(2′-5′)-N2 unit in the antisense strand does not alter the ‘clover leaf’ bend and sugar puckers that are critical for anchoring the 5′-phosphate to Ago 2 MID domain. Importantly, 2′-5′-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2′-5′/3′-5′-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.

Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

“…For strand-nicking cleavage assays utilizing radiolabeled target DNA strands, individual DNA strands (cpEGIPe target sequence) were 5′ radiolabeled with T4 polynucleotide kinase and 32 P-γ-ATP as previously described 112 . Radiolabeled DNA strands were then gel-purified from a 15% denaturing Urea-PAGE by crushand-soak elution after visualizing radioactive bands by autoradiography.…”

Chemical strategies for strand selection in short-interfering RNAs

“…4 ) is well tolerated in the sense strand. 77 Some 2′–5′ phosphate linkages are tolerated on antisense strand, but modification of all six nucleotides from AS:1 to AS:6 reduced activity nearly 8-fold. Positional screening revealed that none of the first six linkages played a substantially larger role than the others and that up to two modifications are relatively well tolerated.…”

Inhaled siRNA Formulations for Respiratory Diseases: From Basic Research to Clinical Application

“…Except in exceptional circumstances, increasing the number of 2 ,5 -phosphodiester bonds reduces siRNA activity, which may be attributed to less siRNA binding to the AGO2 protein. Another study discovered that siRNAs containing a mixture of 2 ,5 /3 ,5 -phosphodiester bonds on the AS were more effective at silencing genes than siRNAs containing only 2 ,5 -phosphodiester bond modifications [93].…”