Protocol for efficient fluorescence 3′ end-labeling of native noncoding RNA domains

Abstract

Noncoding RNAs (ncRNAs) comprise a class of versatile transcripts that are highly involved in the regulation of a wide range of biological processes. Functional long ncRNAs (> 200 nts in length) often adopt secondary structures that arise co-transcriptionally. To maintain the secondary structure elements as well as preparation homogeneity of such transcripts, native-like conditions should be maintained throughout the in vitro synthesis, purification and chemical tagging processes. In this optimized protocol, we describe a simple method for obtaining homogenous samples followed by chemically tagging the 3′ termini of natively-purified structured ncRNA domains that are longer than 200 nts. This protocol replaces traditional hazardous radioactive labeling with fluorescence tagging, and eliminates laborious and time consuming RNA purification and concentration steps and replaces them with straightforward recovery of RNA through centrifugal filtration, preserving the homogeneity and mono-dispersion of the preparations. The protocol provides:•An integrative, simple and straightforward approach for synthesis, purification and labeling of structured ncRNAs whilst maintaining their secondary structure intact.•Replacing hazardous, laborious and time-consuming radioactive labeling of RNA with much simpler fluorescence tagging, thereby facilitating potential downstream applications such as electrophoretic mobility shift assay (EMSA).•A versatile protocol that could be applicable to a wide-range of chemical tags and in principle could be used to label DNA or RNA.