Synthesis of adenosine derivatives as transcription initiators and preparation of 5′ fluorescein- and biotin-labeled RNA through one-step in vitro transcription

Abstract

Expanding our previous finding of an adenosine-initiated transcription system, we now demonstrate that either the 5′ site or the N6 site of adenosine nucleotides can be modified extensively without abolishing their ability to initiate transcription under the T7 [phis]2.5 promoter. Two series of amino derivatives of adenosine nucleotides were synthesized. Fluorescein and biotin groups were coupled to AMP derivatives through linkers of different sizes and hydrophobicities. Both fluorescein- and biotin-conjugated (at either the 5′ or N6 site) adenosine nucleotides can act as efficient transcription initiators, producing fluorescein- and biotin-labeled RNA at the specific 5′ end by a one-step transcription procedure, eliminating posttranscriptional modification. Furthermore, N6-modified adenosine derivative-initiated transcription synthesizes 5′ end modified RNA with a free phosphate group, providing the possibility for further derivatization. The current finding makes easily available a variety of site-specifically functionalized RNA, which may be used in nucleic acid detection, RNA structural and functional investigation, and generation and isolation of novel functional RNA.