Abstract
Arrays of singly labeled 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 (i) all steps of the oligonucleotide labeling—including conjugation, enzymatic synthesis, and product purification—can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.
Highlights
The ability to quantify biomolecules in space and time in living matter has introduced a qualitative change in many fields of biology, allowing the building of accurate mathematical models that can test, validate, and refine hypotheses about biological processes
This method relies on the template-independent addition of dNTPs at the 3′ end of DNA by terminal deoxynucleotidyl transferase (TdT) (Motea and Berdis 2010). This enzyme could elongate 3′ ends indefinitely; the use of dideoxy nucleotides ensures that only a single labeled nucleotide is incorporated into each and almost every oligonucleotide molecule added to the reaction. The feasibility of such a direct labeling strategy was demonstrated previously for individual oligonucleotides (Jahn et al 2011; Winz et al 2015), here we show that it is possible to label sets of conventional PCR oligos using custom-made dye/ biotin conjugated ddUTPs synthesized in a standard biology laboratory (Fig. 1A)
We first tested whether TdT could incorporate unpurified Atto565–ddUTP, synthesized through conjugation of Atto565–NHS ester to NH2ddUTP, into the 3′ end of single DNA oligonucleotides
Summary
The ability to quantify biomolecules in space and time in living matter has introduced a qualitative change in many fields of biology, allowing the building of accurate mathematical models that can test, validate, and refine hypotheses about biological processes. Today a wide variety of RNA detection techniques with single-molecule sensitivity exists (Gaspar and Ephrussi 2015), the pioneering techniques of singlemolecule fluorescent in situ hybridization (smFISH) were based on an array of short oligonucleotides carrying a welldefined number of labels, reducing variance of the detected signal while maintaining a high signal-to-noise ratio (Femino et al 1998; Raj et al 2008). Such arrays typically consist of 24–96 different probes: 18to 22-nucleotide (nt)-long single-stranded DNA (ssDNA) molecules complementary to nonoverlapping segments of the target RNA, each carrying a single label (Raj et al 2008). The relatively large number of probes ensures reliable detection of virtually all transcripts found within the specimen
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