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Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis

Abstract

Methods for fluorescent probing at a defined position of RNA provide powerful tools for analyzing the local structural conformation of functional RNA molecules by tracking fluorescence changes. In this article, we describe the site-specific fluorescent probing of RNA by transcription with an expanded genetic alphabet, using an extra, unnatural base pair between 2-amino-6-(2-thienyl)purine (s) and pyrrole-2-carbaldehyde (Pa). The protocol comprises template DNA preparation containing Pa, transcription involving fluorescent s incorporation and structural analysis of transcripts. The s base is strongly fluorescent, and its nucleoside 5′-triphosphate is site-specifically incorporated into RNA transcripts, opposite Pa in DNA templates, by conventional T7 transcription. The fluorescent intensity of s changes depending on its environment around the probe site, providing clues about the local structural features of RNA molecules. This is the first protocol for RNA transcript preparation with fluorescent labeling at a desired position. The procedure for s-containing RNA preparation takes about 2–3 d.

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Figure 1: The sPa transcription system.
Figure 2
Figure 3: Tertiary structure of tRNAPhe and close-up views around the 47th and 57th bases (PDB ID: 1EVV).
Figure 4: Gel electrophoresis of transcripts generated by the unnatural base-pair system.
Figure 5: The fluorescent intensity of tRNA molecules containing s at the 47th or 57th position.

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Acknowledgements

This work was supported by the Targeted Proteins Research Program and the RIKEN Structural Genomics/Proteomics Initiative, the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant-in-Aid for Scientific Research (KAKENHI 19201046 to I.H., 20710176 to M.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Y.H. conducted most of the experiments and the data analysis; M.K. and I.H. conceived and designed the study, supervised the work and prepared samples; S.Y. supervised the work.

Corresponding authors

Correspondence to Shigeyuki Yokoyama or Ichiro Hirao.

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Competing interests

Authors are employees of TagCyx Biotechnologies or receive funding from TagCyx Biotechnologies.

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Hikida, Y., Kimoto, M., Yokoyama, S. et al. Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis. Nat Protoc 5, 1312–1323 (2010). https://doi.org/10.1038/nprot.2010.77

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