Context-sensitivity of isosteric substitutions of non-Watson–Crick basepairs in recurrent RNA 3D motifs

Abstract

Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson–Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson–Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.

Design and Characterization of Compact, Programmable, Multistranded Nonimmunostimulatory Nucleic Acid Nanoparticles Suitable for Biomedical Applications

“…The UV melting technique is widely employed for the investigation of the thermal stability of nucleic acid-based complexes. ,, All constructs exhibit characteristic single-transition sigmoidal curves, with the calculated melting temperatures (Tm) being remarkably consistent, ranging around 62 °C in CB1 and approximately 56 °C in CB2 as exemplified in Figure C and summarized in Table . Similar to the corresponding d3WJs, the constructs are found to be more thermally stable in the presence of high sodium ions.…”

Toehold-Mediated Shape Transition of Nucleic Acid Nanoparticles

“…PCR products were purified using the QiaQuick PCR purification kit (Qiagen Sciences, Maryland 20874) prior in the vitro transcription reaction. All transcribed RNA products were purified by denaturing 20% polyacrylamide gel (29:1 acrylamide/bisacrylamide) electrophoresis (PAGE) containing 8 M UREA …”

“…All transcribed RNA products were purified by denaturing 20% polyacrylamide gel (29:1 acrylamide/bisacrylamide) electrophoresis (PAGE) containing 8 M UREA. 15 The co-transcriptional synthesis of the RNA hexagonal complex was achieved by introducing seven corresponding purified PCR DNA templates into an in vitro T7 RNA transcription mixture, with equal ratios. After 4 hours of incubation at 37 °C, DNase I treatment was applied to remove the DNA sequences, and the resulting crude RNA product was analyzed using gel shift assay and fluorescence study without purification.…”

“…Nanoparticles can be fabricated from various materials ranging from metal atoms (e.g., gold, silver, and iron oxide nanoparticles), − lipid-based materials (e.g., micelles), and amino acids (e.g., antibodies and vaccines) − to nucleic acids (e.g., nanostructures made of DNA or RNA) or their chemical analogues. , While all these nanomaterials were shown to be effective in synthesis and self-assembly of functional nanoscale-sized objects, nucleic acids offer multiple unique advantages. − The ability of nucleic acids, in particular RNA, to form both canonical (cis-Watson–Crick) and non-canonical base pairs , gives rise to a library of structural motifs with a myriad of architectural differences. − This library is often viewed as modular building blocks to fabricate nucleic acid nanoparticles (NANPs) and nanoscale DNA assemblies in a controlled and pre-programmed fashion. − In addition, the secondary structures of both DNA and RNA can be predicted with high level of accuracy, thus opening possibilities to generate modular NANPs with defined thermodynamic properties and the ability to dynamically respond to the external stimuli. − The intrinsic properties of nucleic acids to form non-covalent interactions with small molecules, metal ions, polypeptides, other nucleic acids, and large proteins enable the possibility to fabricate sophisticated nanodevices using DNA and RNA as building materials. − These unique features expand the possibility of NANPs to serve as highly programmable materials, suitable for applications in synthetic biology, biotechnology, and biosensing.…”

Studies on Viroid Shed Light on the Role of RNA Three-Dimensional Structural Motifs in RNA Trafficking in Plants

“…In contrast, loop regions are composed of diverse non-WC base pairs that are highly arranged ( Wang et al, 2018 ). Many loop motifs can be found at nonhomologous positions of diverse RNA species but largely keep the base pair geometries and interaction details, so they are also termed recurring loop motifs ( Khisamutdinov et al, 2021 ). RNA 3D loop motifs provide recognition sites for specific RNA-protein, RNA–RNA, and RNA-ligand interactions.…”