Assessment and comparison of thermal stability of phosphorothioate-DNA, DNA, RNA, 2′-F RNA, and LNA in the context of Phi29 pRNA 3WJ

Abstract

The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, 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 the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. 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 RNA < 2'-F RNA/2'-F RNA < 2'-F RNA/LNA < LNA/LNA. This proposition is supported by 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 from 21.2°C to over 95°C. The 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 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 their pharmacokinetics profile.

The immunorecognition, subcellular compartmentalization, and physicochemical properties of nucleic acid nanoparticles can be controlled by composition modification

“…In agreement with our previous works, complete dissociation tended to occur at higher temperatures for NANPs with a central RNA strand ( 3 , 6 , 35 ). Consistent with previous findings, incorporation of 2′F modified strands resulted in higher T m s, with the fully modified NANPs displaying the highest T m due to better stacking between adjacent base-pairs within the resulting secondary structure ( 36 , 37 ).…”

Multi-targeted Antisense Oligonucleotide Delivery by a Framework Nucleic Acid for Inhibiting Biofilm Formation and Virulence

“…Based on this information, we designed the antisense oligonucleotide sequence DTWACANNNNND-TAACA (where D is A, G, or T and W is A or T) to target the conserved sequences of the gtfBCD, gbpB, and ftf promoter regions [32]. Because pure DNA or RNA is susceptible to nuclease degradation, we designed a sequence of nested antisense oligonucleotides made of DNA and RNA, with DNA modified by phosphorothioate and RNA by 2′ O-Me [35][36][37].…”

High-order structures from nucleic acids for biomedical applications

“…97,98 Recent work has narrowed engineering efforts to the unusually stable 3WJ motif found within the pRNA structure. [99][100][101] The phi29 pRNA 3WJ has been used to construct RNA polygons, 102-105 tetrahedra 106 (Fig. 4E), and prisms.…”