Synthesis and Biological Activity of Short Interfering RNAs Having Several Consecutive Amide Internucleoside Linkages

Abstract

The successo fR NA interference (RNAi)a saresearch tool and potential therapeutic approachh as reinvigorated interesti nc hemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amidesm ay be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) criticalf or in vivo applications.I nt his study,w ei ntroduced up to seven consecutive amide linkages at the 3'-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutivea mide linkages retained high RNAi activity when paired with ap assenger strandhavingo ne amide modifica-tion between its first and second nucleosidesa tthe 5'-end. Further increasei nt he number of modifications decreased the RNAia ctivity;h owever,s iRNAs with sixa nd seven amide linkages still showedu seful target silencing. While an siRNA duplexh aving nine amide linkages retained somes ilencing activity,t he partial reduction of the negative charged id not enable passiveu ptake in HeLa cells. Our resultss uggest that further chemical modifications, in addition to amide linkages, are neededt oe nablec ellular uptake of siRNAs in the absence of transfection agents.[a] Dr.Scheme1.Synthesis of monomers 3 and 4.Steps:a)TOM-Cl, Bu 2 SnCl 2 , DIPEA,C lCH 2 CH 2 Cl, reflux,1h, 26 %o f5 and 41 %o f6;b)H 2 S( gas), pyridine/water(4:1), RT,overnight;c )p-methoxytrityl chloride,D MAP,pyridine, RT,overnight, 65 %o f7 and 65 %o f8;d)succinic anhydride, DBU, CH 2 Cl 2 , RT,30min, 80 %; e) P(OCH 2 CH 2 CN)(N(iPr) 2 ) 2 ,t etrazole, N-methylimidazole, CH 2 Cl 2 ,RT, 24 h, 35 %.

Optimization of Automated Synthesis of Amide-Linked RNA

“…Using the optimized automated solid-phase protocol, we synthesized a 21-nucleotide-long siRNA guide strand having six consecutive amide linkages at the 3′-end with an overall yield of ∼1%, which was a significant improvement compared to the 0.14% yield achieved using our previously published manual procedures. 13 Our results suggested that the main limitation of coupling efficiency was steric hindrance, originating from both bulky 2′-O-TBS and steric crowding on the solid support. These findings will facilitate future studies on AM1 modifications in functional RNAs, such as siRNAs and CRISPR RNAs.…”

“…Succinate ester 3 ( 13 ) and monomer 2b ( 17 ) were synthesized as previously reported. Monomer 2c was synthesized from the known intermediate 4 ( 6 ) following our previously developed procedures ( Scheme 1 ).…”

“… 13 Placing several consecutive AM1 linkages in siRNAs decreased the RNAi activity; however, detailed studies on such siRNAs were hampered by lengthy manual procedures and poor yields of solid-phase amide couplings. 8 , 13 …”

“… 8 Using HATU or PyAOP activation, coupling yields of 2b did not exceed 80–85% using double couplings (a 4 h coupling followed by a 12 h coupling), which allowed us to synthesize an amide-linked RNA having seven consecutive amide linkages, albeit with poor overall yield. 13 Collectively, these studies revealed that the yield of amide coupling was inferior to that of >95% expected in solid-phase RNA phosphoramidite synthesis, and taken together with long coupling times, it was the limiting factor for studies on amide-linked RNA.…”

Adaptormoleküle zur Rekrutierung von Transkriptionsfaktoren oder miRNAs an nicht native Bindestellen

“…RNA-Interferenz (RNAi) ist ein komplexer Prozess, der hautsächlich zur Abwehr von Virusinfektionen in einer Zelle dient. [167][168][169] [167] Man spricht vom RNA-induced silencing complex (RISC). Diese…”