Site-Specific Dual-Color Labeling of Long RNAs

Labeling of large RNAs with reporting entities, for example, fluorophores, has significant impact on RNA studies in vitro and in vivo. Here we describe a minimally invasive RNA labeling method featuring nucleotide- and position-selectivity, which solves the long-standing challenge of how to achieve accurate site-specific labeling of large RNAs with a least possible influence on folding and/or function. We use a custom-designed reactive DNA strand to hybridize to the RNA and transfer the alkyne group onto the targeted adenine or cytosine. Simultaneously, the 3'-terminus of RNA is converted to a di-aldehyde moiety under the experimental condition applied. The incorporated functionalities at the internal and the 3'-terminal sites can then be conjugated with reporting entities via bioorthogonal chemistry. This method is particularly valuable for, but not limited to, single-molecule fluorescence applications. We demonstrate the method on an RNA construct of 275 nucleotides, the btuB riboswitch of Escherichia coli.