6533b81ffe1ef96bd1278573
RESEARCH PRODUCT
Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling.
Roman TeimerKaloian KoynovSophie WillnowElmar WeinholdJürgen BurhenneMark HelmYuri Motorinsubject
S-AdenosylmethioninetRNA MethyltransferasesBase SequenceStereochemistryMolecular Sequence DataGuanosineRNAFluorescence correlation spectroscopyBiologyTRNA Methyltransferaseschemistry.chemical_compoundRNA Transfer PheSpectrometry FluorescencechemistryBiochemistryAlkynesTransfer RNASynthetic Biology and ChemistryGeneticsClick chemistryMoietyClick ChemistryAzideOrganic ChemicalsFluorescent Dyesdescription
This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2010-11-02 | Nucleic acids research |