Combination of chemical and enzymatic RNA synthesis.
The potential of standard in vitro transcription reactions can be dramatically expanded, if chemically synthesized low-mol-wt compounds are used as building blocks in combination with standard nucleotide 5' triphosphates (NTPs). Short oligonucleotides that terminate in guanosine effectively compete with guanosine 5' triphosphate (GTP) as starter building blocks, and they are incorporated at the 5'-end of transcripts. Applications include production of RNAs with "unfriendly 5'-ends" (they do not begin with G), variations of the 5'-sequence are possible with the same DNA template, site-specific insertion of nucleotide modifications, and addition of 5'-labels, such as fluorescein for detection or biotin for capture. Clearly, chemically synthesized, modified NTPs are inserted at internal sites. The combination with phosphorothioate linkages for detection has been developed into a powerful high-throughput method to study site-specific interference of modifications with RNA function.
Gaur
Rajesh K
RK
Graduate School of Biological Sciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA.
Hanne
Andreas
A
Krupp
Guido
G
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis* / Q000138:chemical synthesis*; D012343:RNA, Transfer / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D007202:Indicators and Reagents; D012313:RNA; D012343:RNA, Transfer; D012321:DNA-Directed RNA Polymerases