Enzymatic production of single molecule FISH and RNA capture probes

Abstract

AbstractArrays of singly-labelled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single molecule microscopy analysis and high efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that 1) all steps of the oligonucleotide labelling – including conjugation, enzymatic synthesis and product purification – can be performed in a standard biology laboratory, 2) the process yields >90 %, often >95 % labeled product with minimal carry-over of impurities and 3) the oligonucleotides can be labeled with different dyes or biotin, allowing single molecule FISH or RNA affinity purification to be performed.Graphical abstract