Nonradioactive labeling of RNA.
A modified ribodinucleotide (named pSEEp) has been synthesized using commercially available components on a DNA synthesizer. The presence of a 3'-terminal primary amino group permits its coupling to a range of nonradioactive labels, exemplified here by fluorescein. The product of chemical derivatization of the parent dinucleotide is a good substrate for T4 RNA ligase-mediated coupling to RNA under very mild conditions; the target RNA is not itself subjected to chemical treatment. The well-defined product of this terminal labeling may be analyzed by, for example, the use of a fluorescence-based DNA sequencer. The applications documented serve to demonstrate the power of this approach suggesting that any procedure depending on the detection or targeted tagging of RNA may be adapted to using pSEEp and its derivatives.
Igloi
G L
GL
Institut für Biologie III, Universität Freiburg, Germany.
D019793:Fluorescein; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D009841:Oligonucleotides; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry*; D011130:RNA Ligase (ATP) / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000032:analysis; D012365:RNA, Transfer, Tyr / Q000032:analysis; D013379:Substrate Specificity
D005452:Fluoresceins; D007202:Indicators and Reagents; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; C099647:cytidylyl-(3'-5')-3'-O-(3-N-((3-carboxy-4-(3,6-dihydroxy-9H-xanthen-9-yl)phen-2-yl)carbonyl)amino-2-hydroxypropyl)cytidylic acid; C099646:cytidylyl-(3'-5')-3'-O-(3-amino-2-hydroxypropyl)cytidylic acid; D012313:RNA; D011130:RNA Ligase (ATP); D019793:Fluorescein