Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription.
Nucleic Acids Res, 2003/2/01;31(3):e8.
Huang F[1]
Affiliations
PMID: 12560511
Impact factor: 19.16
Abstract
Protein enzymes frequently recruit small molecule coenzymes to perform a variety of biochemical reactions. While the catalytic activities of RNA have been expanding rapidly, a similar strategy for RNA to utilize coenzymes and to increase its functional capabilities has yet to be demonstrated. A general in vitro transcription procedure has been developed to efficiently prepare RNA with coenzymes CoA, NAD and FAD covalently attached to the 5' end. These adenosine-containing coenzymes initiate transcription under the T7 class II promoter by T7 RNA polymerase. In addition to the three coenzymes, other adenosine-containing molecules may be incorporated into the first nucleotide position of RNA as well. This method provides easy access to CoA-, NAD- and FAD-RNA, which may find broad applications in generating coenzyme- utilizing ribozymes. In addition, both oxidized FAD and reduced NADH are highly fluorescent. NADH-RNA and FAD-RNA can therefore be used as probes for DNA/RNA detection and for structural investigation of RNA function by fluorescence spectroscopy.
MeSH terms
Adenosine; Base Sequence; Coenzyme A; Flavin-Adenine Dinucleotide; Molecular Sequence Data; NAD; RNA; Spectrometry, Fluorescence; Transcription, Genetic
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