Synthesis, Gene Silencing, and Molecular Modeling Studies of 4′-C-Aminomethyl-2′-O-methyl Modified Small Interfering RNAs

Abstract

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (∼1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.

Incorporation of 4′-C-aminomethyl-2′-O-methylthymidine into DNA by thermophilic DNA polymerases

“…9 We have recently demonstrated advantageous properties of the corresponding 4 0 -C-aminomethyl-2 0 -O-methyl-modified uridine and cytidine nucleosides to enhance the therapeutic potential of small interfering RNAs (siRNA). 10 Inspired by these results, we seek to select DNA enzymes that contain amm-modified nucleotides in the catalytic core. The analogously modified thymidine has been previously investigated as modification of antisense oligonucleotides, 11 but the corresponding triphosphate and its enzymatic recognition have not been reported.…”

4′-Guanidinium-modified siRNA: a molecular tool to control RNAi activity through RISC priming and selective antisense strand loading

“…The 5 0 hydroxyl of compound 10 was selectively protected using 4,4 0 -dimethoxytrityl chloride (DMTr-Cl) to furnish 11. 22 Finally, phosphitylation of compound 11 was carried out to furnish the phosphoramidite 12. 22 The unmodified and GMU modified siRNAs targeting signal transducer and activator of transcription 3 (STAT3) mRNA were synthesized using a solid phase RNA synthesizer ( Table 1).…”

Development of Therapeutic-Grade Small Interfering RNAs by Chemical Engineering

“…PACT and ThioPACT enhance siRNA stability, and will reduce siRNA potency, even if only placed in the passenger strand, and (double-stranded) siRNA are not efficiently taken up by cells in the absence of transfection reagents, in contrast to modified single-stranded oligonucleotides (Threlfall et al, 2012). Finally, 4′-aminomethyl-2′-OMe dual modification of siRNAs will slightly lower the T m (∼1°C/modification) compared to the unmodified duplex, can increase siRNA serum stability and modifications are well-tolerated at various positions in the passenger strand, but not in the seed region of the guide strand (Gore et al, 2012). …”