Integrating PLOR and SPAAC Click Chemistry for Efficient Site-Specific Fluorescent Labeling of RNA DOI Open Access
Yanyan Xue, Si Xiao,

Daxu Yin

и другие.

International Journal of Molecular Sciences, Год журнала: 2025, Номер 26(6), С. 2601 - 2601

Опубликована: Март 13, 2025

Precisely fluorescently labeling specific nucleotide sites of RNA is critical for gaining insights into the structure and function through multiple fluorescence detection techniques. The position-selective (PLOR) method provides a promising strategy to achieve this, wherein fluorophore-modified NTPs can be co-transcriptionally introduced nascent by using T7 polymerase (T7 RNAP). However, due steric hindrance limitations, efficiency RNAP in recognizing incorporating large far from satisfactory. To overcome this challenge, work, we developed an efficient PLOR variant (ePLOR) site-specific fluorescent integrating with post-transcriptional SPAAC (strain-promoted azido-alkyne cycloaddition) click chemistry reaction. reaction occurring on nearly 100%. Consequently, ePLOR enables precise designated across various structural regions SAM-VI riboswitch adenine RNA, synthesis efficiencies that are 2–2.5 times higher than those PLOR. work used broader spectrum long-strand RNAs greatly facilitate these RNAs.

Язык: Английский

Integrating PLOR and SPAAC Click Chemistry for Efficient Site-Specific Fluorescent Labeling of RNA DOI Open Access
Yanyan Xue, Si Xiao,

Daxu Yin

и другие.

International Journal of Molecular Sciences, Год журнала: 2025, Номер 26(6), С. 2601 - 2601

Опубликована: Март 13, 2025

Precisely fluorescently labeling specific nucleotide sites of RNA is critical for gaining insights into the structure and function through multiple fluorescence detection techniques. The position-selective (PLOR) method provides a promising strategy to achieve this, wherein fluorophore-modified NTPs can be co-transcriptionally introduced nascent by using T7 polymerase (T7 RNAP). However, due steric hindrance limitations, efficiency RNAP in recognizing incorporating large far from satisfactory. To overcome this challenge, work, we developed an efficient PLOR variant (ePLOR) site-specific fluorescent integrating with post-transcriptional SPAAC (strain-promoted azido-alkyne cycloaddition) click chemistry reaction. reaction occurring on nearly 100%. Consequently, ePLOR enables precise designated across various structural regions SAM-VI riboswitch adenine RNA, synthesis efficiencies that are 2–2.5 times higher than those PLOR. work used broader spectrum long-strand RNAs greatly facilitate these RNAs.

Язык: Английский

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