Abstract
Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.
Highlights
RNA molecules have enormous versatility within living organisms
Chemical synthesis is limited by the length of the RNA molecule, and posttranscriptional modifications are applicable only to the sitespecific 5 - or 3 -terminal labeling of transcripts
High resolution mass spectra (HRMS) and electrospray ionization mass spectra (ESIMS) were recorded on a Varian 901-MS spectrometer and a Waters micromass ZMD 4000 mass detector equipped with a Waters 2690 LC system, respectively
Summary
Structural and biological studies of functional RNA molecules will be facilitated by the site-specific labeling and probing of target RNAs without the loss of activity. The present methods for the chemical synthesis of labeled RNA molecules or post-transcriptional modifications of RNA are very restrictive. Chemical synthesis is limited by the length of the RNA molecule, and posttranscriptional modifications are applicable only to the sitespecific 5 - or 3 -terminal labeling of transcripts. Progress in click chemistry has been increasing the feasibility of RNA modifications (1–8). Copper-free click chemistry between an azide compound and a cyclooctyne reagent is becoming very popular (9–12). A method to embed an azide component into RNA molecules at desired positions could facilitate various modifications with any probes, and contributing to functional RNA studies
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