Combination of chemical and enzymatic RNA synthesis.

Abstract

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.