Talk on Saturday 10:45-11:00am submitted by Xijun Piao
Assessment of thermal stability of phosphorothioate-DNA, DNA, RNA, 2’-F RNA and LNA in the context of Phi29 pRNA 3WJ
Xijun Piao (College of Pharmacy, The Ohio State University), Hongzhi Wang (College of Pharmacy, The Ohio State University), Daniel W. Binzel (College of Pharmacy, The Ohio State University), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy; College of Medicine; Dorothy M. Davis Heart and Lung Research Institute; James Comprehensive Cancer Center, The Ohio State Univ.)
Thermodynamically-stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2’-F nucleotides (2’-F) and locked nucleic acids (LNA) on the thermal and in vivo stability of RNA and the three-way junction (3WJ) of bacteriophage phi29 motor pRNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2’-F < 2’-F/2’-F < 2’-F/LNA < LNA/LNA. This proposition is supported by the studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically-modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures (Tms) from 21.2 oC to over 95 oC. The LNA 3WJ was resistant to boiling temperature denaturation, urea denaturation and 50% serum degradation. Intravenous injection of fluorescent LNA/2’-F hybrid 3WJs into mice revealed its exceptional in vivo stability and the presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2’-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve its pharmacokinetics profile.
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(4) Piao X, Wang H, Binzel DW, Guo P. in revision, 2017.
Keywords: Three-way junction, thermal stability, in vivo stability