Efficient DNA fluorescence labeling via base excision trapping

Yong Woong Jun, Emily M. Harcourt, Lu Xiao, David L. Wilson and Eric T. Kool ()
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Yong Woong Jun: Stanford University
Emily M. Harcourt: Le Moyne College
Lu Xiao: Stanford University
David L. Wilson: Stanford University
Eric T. Kool: Stanford University

Nature Communications, 2022, vol. 13, issue 1, 1-10

Abstract: Abstract Fluorescence labeling of DNAs is broadly useful, but methods for labeling are expensive and labor-intensive. Here we describe a general method for fluorescence labeling of oligonucleotides readily and cost-efficiently via base excision trapping (BETr), employing deaminated DNA bases to mark label positions, which are excised by base excision repair enzymes generating AP sites. Specially designed aminooxy-substituted rotor dyes trap the AP sites, yielding high emission intensities. BETr is orthogonal to DNA synthesis by polymerases, enabling multi-uracil incorporation into an amplicon and in situ BETr labeling without washing. BETr also enables labeling of dsDNA such as genomic DNA at a high labeling density in a single tube by use of nick translation. Use of two different deaminated bases facilitates two-color site-specific labeling. Use of a multi-labeled DNA construct as a bright fluorescence tag is demonstrated through the conjugation to an antibody for imaging proteins. Finally, double-strand selectivity of a repair enzyme is harnessed in sensitive reporting on the presence of a target DNA or RNA in a mixture with isothermal turnover and single nucleotide specificity. Overall, the results document a convenient and versatile method for general fluorescence labeling of DNAs.

Date: 2022
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DOI: 10.1038/s41467-022-32494-8

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