Novel cyanine-AMP conjugates for efficient 5′ RNA fluorescent labeling by one-step transcription and replacement of [γ-32P]ATP in RNA structural investigation

Abstract

Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 φ2.5 promoter. Efficient fluorophore labeling of 5′ RNA is achieved in a single transcription step by including F550/570 and F650/ 670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [γ-32P]ATP to prepare 5′ labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [γ-32P]ATP. © The Author 2005. Published by Oxford University Press. All rights reserved.