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Nature Communications · 2021 ·Vol. 12 ·期 1 ·1-14 · IF 14.919

Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

Wang Yuqin,Guan Xiaoyu,Zhang Shanyu,Liu Yao,Wang Sha,Fan Pingping,Du Xiaoyu,Yan Shuanghong,Zhang Panke,Chen Hong-Yuan,Li Wenfei,Zhang Daoqiang,Huang Shuo

Folding of RNA can produce elaborate tertiary structures, corresponding to their diverse roles in the regulation of biological activities. Direct observation of RNA structures at high resolution in their native form however remains a challenge. The large vestibule and the narrow constriction of a Mycobacterium smegmatis porin A (MspA) suggests a sensing mode called nanopore trapping/translocation, which clearly distinguishes between microRNA, small interfering RNA (siRNA), transfer RNA (tRNA) and 5 S ribosomal RNA (rRNA). To further profit from the acquired event characteristics, a custom machine learning algorithm is developed. Events from measurements with a mixture of RNA analytes can be automatically classified, reporting a general accuracy of ~93.4%. tRNAs, which possess a unique tertiary structure, report a highly distinguishable sensing feature, different from all other RNA types tested in this study. With this strategy, tRNAs from different sources are measured and a high structural conservation across different species is observed in single molecule.

研究领域: 纳米技术 · 生物物理学 · 生物信息学 · 分子生物学

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Current Protocols · 2024 ·Vol. 4 ·期 1 ·e956 · IF 0

Synthesis of Nucleotides Bearing the 2′-O-Trifluoromethyl Group and Their Application in RNA Analogs Preparation

Xuan Chen,Long Xie,Chenglong Zhang,Shen Tian,Zeyu Tang,Ming Xiang,Weiwei Tian,Patrick Lu,Xianbin Yang

The integration of fluorine atoms into biologically active organic compounds has proved to be a vital technique in small molecule drugs. This technique can substantially enhance crucial properties, including metabolic stability, lipophilicity, and bioavailability, often with a mere addition of a single fluorine atom or a trifluoromethyl group. Over the past few decades, this concept has also been applied in nucleic acid chemistry. A commonly employed 2′-OH substitution is the introduction of a 2′-deoxy-2′-fluoro (2′-F) group. The strong electronegativity of fluorine prompts the modified siRNA to readily adopt a C3′- endo conformation, resulting in significant advantages in terms of binding affinity. To enrich the toolbox of chemical modification of oligonucleotides, the replacement of the 2′-OH with the 2′- O -trifluoromethyl group has been developed in RNA analog synthesis. Oligodeoxynucleotides containing the 2′- O -trifluoromethyl group can greatly increase the thermal stability of DNA/RNA duplexes depending on the position and amount of the modification. Moreover, 2′- O -trifluoromethylated oligodeoxynucleotide also exhibited a slightly higher resistance to snake venom phosphodiesterase than the unmodified oligodeoxynucleotide. The 2′- O -trifluoromethylated oligonucleotides can emerge as a label to study RNA structure and function as well, or to develop DNA/RNA-based diagnostics. Hence, it is necessary to report an effective method for the synthesis, deprotection, purification, and characterization of oligonucleotides bearing a 2′ -O -trifluoromethyl group. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Preparation of 6- N -benzoyl-5′- O -dimethoxytrityl-2′- O -trifluoromethyl adenosine 3′-(2-cyanoethyl N,N -diisopropyl)phosphoramidite Basic Protocol 2 : Preparation of 4- N -acetyl-5′- O -dimethoxytrityl-2′- O -trifluoromethyl cytidine 3′-(2-cyanoethyl N,N -diisopropyl)phosphoramidite Basic Protocol 3 : Preparation of 2- N -isobutyryl-5′- O -dimethoxytrityl-2′- O -trifluoromethyl guanine 3′-(2-cyanoethyl N,N -diisopropyl)phosphoramidite Basic Protocol 4 : Preparation of 5′- O -dimethoxytrityl-2′- O -2-trifluoromethyl uridine 3′-(2-cyanoethyl N,N -diisopropyl) phosphoramidite Basic Protocol 5 : Solid-phase synthesis of 2′- O -trifluoromethylated RNA analogs Basic Protocol 6 : Deprotection and purification of 2′- O -trifluoromethyl-RNAs

研究领域: 生物有机化学 · 核酸化学 · 药物化学 · RNA生物学 · 分子生物学 · 药学

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Structural-profiling of low molecular weight RNAs by nanopore trapping/translocation using Mycobacterium smegmatis porin A

Synthesis of Nucleotides Bearing the 2′-O-Trifluoromethyl Group and Their Application in RNA Analogs Preparation