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. 2007;35(16):5360-9.
doi: 10.1093/nar/gkm508. Epub 2007 Aug 9.

Fluorescent probing for RNA molecules by an unnatural base-pair system

Michiko Kimoto  1 , Tsuneo Mitsui, Yoko Harada, Akira Sato, Shigeyuki Yokoyama, Ichiro Hirao
Affiliations

Fluorescent probing for RNA molecules by an unnatural base-pair system

Michiko Kimoto et al. Nucleic Acids Res. 2007.

Abstract

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5'-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions.

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Figures

Figure 1.
Figure 1.
The unnatural base-pair system for specific replication and transcription. (A) Structures of the unnatural s–Pa, Ds–Pa, s–z and s′–Pa pairs. (B) The unnatural base-pair system for site-specific incorporation of the fluorescent s base into RNA by transcription using the Pa-containing DNA templates, which can be amplified by PCR mediated by the Ds–Pa pair.
Figure 2.
Figure 2.
T7 transcription mediated by the s–Pa pairing. (A) Schemes of the experiments. (B) Gel electrophoresis of transcripts generated from templates containing one or two Pa or z bases with the natural NTPs (1 mM) and sTP (1 mM). Transcripts were labeled at their 5′-termini with [γ-32P]GTP. The relative yields of each transcript were determined by comparison to the yields of native transcripts from templates consisting of the natural bases, and each yield was averaged from 3 to 4 data sets. (C) 2D-TLC analysis of the labeled ribonucleoside 3′-phosphates obtained from the nuclease digestion of the transcripts (17-mer). The transcripts were internally labeled with [α-32P]UTP. The spots on the TLC were obtained from the 17-mer fragment transcribed from the template (N1N2N3 = PaAC or CAC) in the presence of 1 mM sTP.
Figure 3.
Figure 3.
Incorporation of the fluorescent s base into GNRA hairpins. (A) The secondary structure of the RNA hairpin with a GAAA loop. The second A and third A in the loop are shown in blue and red, respectively. (B) The 3D structure of the GAAA-loop hairpin (35). (C) The profiles of the fluorescent intensity at 434 nm (solid lines) and the UV absorption at 260 nm (dotted lines) of the RNA hairpin with a GsAA (RNA hairpin 10s) or GAsA (RNA hairpin 11s) loop. Tm and Tmf values were obtained from the UV melting and fluorescent intensity profiles, respectively.
Figure 4.
Figure 4.
The s incorporation sites in yeast tRNAPhe and the structure of the tRNA. (A) The secondary structure of the original tRNA transcript. The positions substituted with s are circled. The broken lines show base–base interactions for the 3D structure (37). The boxed G–C pair was changed from the original C–G pair, but this mutation does not significantly alter the original tRNA structure. (B–E) The deep-colored bases were substituted with s, which stacks with the light-colored bases, and the yellow spheres represent Mg2+.
Figure 5.
Figure 5.
The fluorescent intensity and UV absorption profiles of tRNA molecules containing s at specific positions. Melting curves obtained by the changes in the fluorescent intensities at 434 nm (solid lines) and in the UV absorbance at 260 nm (dotted lines) of the tRNAs containing s at various positions.
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