Synthesis of biotin-AMP conjugate for 5' biotin labeling of RNA through one-step in vitro transcription.
Biotin-labeled RNA has found broad applications in chemistry, biology and biomedicine. In this protocol, we describe a simple procedure for 5' RNA biotin labeling by one-step in vitro transcription. A biotin-AMP (adenosine 5'-monophosphate) conjugate, biotin-HDAAMP (adenosine 5'-(6-aminohexyl) phosphoramide; where HDA is 1,6-hexanediamine), is chemically synthesized. Transcription initiation by biotin-HDAAMP under the T7 phi 2.5 promoter produces 5' biotin-labeled RNA with high labeling efficiency. The procedure is especially useful for biotin labeling of RNA that is larger than 60 nucleotides. In addition, the protocol provides an attractive alternative to chemical synthesis of biotin-labeled small RNA of less than 60 nucleotides, particularly when the desired quantity of RNA is low. The whole procedure, from chemical syntheses to isolated biotin-labeled RNA, can be completed within 2 weeks.
Huang
Faqing
F
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 39406, USA. faqing.h.huang@usm.edu
He
Jun
J
Zhang
Yilin
Y
Guo
Yanlin
Y
D000249:Adenosine Monophosphate / Q000737:chemistry* / Q000302:isolation & purification; D017123:Bacteriophage T7 / Q000235:genetics; D001710:Biotin / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D019921:Biotinylation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification; D014158:Transcription, Genetic
D000249:Adenosine Monophosphate; D012313:RNA; D001710:Biotin; D004247:DNA