Efficient incorporation of CoA, NAD and FAD into RNA by in vitro transcription.
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.
Huang
Faqing
F
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406-5043, USA. faqing.h.huang@usm.edu
D000241:Adenosine / Q000031:analogs & derivatives; D001483:Base Sequence; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D012313:RNA; D000241:Adenosine; D003065:Coenzyme A