Amides as Excellent Mimics of Phosphate Linkages in RNA

Abstract

Since the discovery that RNA can catalyze chemical reactions, the number and variety of noncoding RNAs and the important roles they play in biology have been growing steadily. Backbone-modified RNA may find broad application in the fundamental biology and biomedicine of noncoding RNAs, providing that the modifications mimic the structure of the phosphodiester linkage and do not alter the conformation of RNA. In particular, the potential of RNA interference to become a new therapeutic strategy has revitalized interest in chemical modifications that may optimize the pharmacological properties of short interfering RNAs (siRNAs).[1] We are interested in hydrophobic nonionic mimics of the phosphate backbone, such as formacetals [2] and amides, [3] that may confer high nuclease resistance to siRNAs along with reduced charge and increased hydrophobicity. Earlier studies showed that 3'-CH 2 -CO-NH-5' internucleoside amide linkages (abbreviated here as AM1) were well-tolerated in the DNA strand of an A-type DNA-RNA heteroduplex.[4] Subsequently, we found that AM1 modifications did not change the thermal stability of RNA-RNA duplexes.[3] Most importantly, Iwase et al. [5] recently showed that AM1 amides were well-tolerated in the 3' overhangs of siRNAs.Taken together, these data suggest that amides may be good mimics of phosphate linkages in RNA; however, beyond simple melting-temperature measurements, the structural and thermodynamic properties of amide-modified RNA have not been established. Herein we present the first comprehensive structural and thermodynamic study that clearly shows that AM1 linkages do not disturb the A-type structure, thermal stability, and hydration of RNA duplexes. Despite the different geometry, amide AM1 appears to be an excellent mimic of the phosphate linkage in RNA. Our study complements structural studies on amide-modified DNA [4,6] and provides the first detailed insight into how the AM1 amide is accommodated in an RNA duplex.We started by designing a new route for the synthesis of the r(U AM1 A) dimer phosphoramidite, which was used to prepare the amide-modified RNA sequences (Scheme 1). The tert-butyldimethylsilyl (TBS) groups in the known 3'-allyluridine 1 [3] were replaced with 5'-O-methoxytrityl (MMT) and 2'-O-acetyl protecting groups suitable for solid-phase RNA synthesis. Two-step oxidative degradation of the alkene gave the carboxylic acid part 6 of the r(U AM1 A) dimer. [4a,b] For the synthesis of the amine part, we designed a novel route involving selective protection of the 2'-OH group of 5'-aminoadenosine with the triisopropylsilyloxymethyl (TOM) group. Treatment of 5'-azido-N-benzoyladenosine (7) with dibutyltin chloride followed by TOM chloride gave a mixture of 2'-and 3'-O-TOM nucleosides, from which the desired compound 8 was isolated in 30 % yield. Reduction of the azide gave the amine 9, which was coupled with the carboxylic acid 6 to give the dimer 10 (Scheme 2). Although protection of the 2'-OH group of adenosine 7 was relatively low-yielding, this strategy was advant...

Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs

“…Consistent with our previous osmotic stress data (23), amide linkages are well hydrated by tandem waters linking the carbonyl group and phosphate oxygens (Figure 4B). The amide conformation in the present structure is similar to that observed in our earlier NMR structure (23).…”

Therapeutic potential of chemically modified siRNA: Recent trends

“…The internucleotide phosphodiester linkages of unmodified siRNAs were negatively charged at physiological pH and can be cleaved by endo‐ and exonucleases. Chemical modifications of phosphodiester linkages with amide modification ( 16 ) using U AM1 U and U AM1 A had been investigated by our group (selvam et al . & Mutisya et al …”

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs

“…Our hypothesis was based on earlier work ( 26 – 31 ) showing that isolated AM1 linkages were well accommodated in DNA–RNA heteroduplexes and all-RNA duplexes, with relatively small changes in thermal stability and overall structures of the modified duplexes. Later, we reported that isolated AM1 linkages were indeed surprisingly good structural mimics of phosphate and did not change the A-type conformation, thermal stability and hydration of short RNA duplexes ( 32 , 33 ). While three consecutive amide linkages in the middle of a short RNA duplex caused some loss of thermal stability, the overall RNA structure was only minimally altered ( 34 ).…”