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Sequence-Specific Post-Synthetic Oligonucleotide Labeling for Single-Molecule Fluorescence Applications
journal contribution
posted on 2016-07-13, 00:00 authored by David Egloff, Igor A. Oleinich, Meng Zhao, Sebastian L. B. König, Roland K. O. Sigel, Eva FreisingerThe sequence-specific
fluorescence labeling of nucleic acids is
a prerequisite for various methods including single-molecule Förster
resonance energy transfer (smFRET) for the detailed study of nucleic
acid folding and function. Such nucleic acid derivatives are commonly
obtained by solid-phase methods; however, yields decrease rapidly
with increasing length and restrict the practicability of this approach
for long strands. Here, we report a new labeling strategy for the
postsynthetic incorporation of a bioorthogonal group into single stranded
regions of both DNA and RNA of unrestricted length. A 12-alkyne-etheno-adenine
modification is sequence-selectively formed using DNA-templated synthesis,
followed by conjugation of the fluorophore Cy3 via a copper-catalyzed azide–alkyne cycloaddition (CuAAC). Evaluation
of the labeled strands in smFRET measurements shows that the strategy
developed here has the potential to be used for the study of long
functional nucleic acids by (single-molecule) fluorescence or other
methods. To prove the universal use of the method, its application
was successfully extended to the labeling of a short RNA single strand.
As a proof-of-concept, also the labeling of a large RNA molecule in
form of a 633 nucleotide long construct derived from the Saccharomyces
cerevisiae group II intron Sc.ai5γ
was performed, and covalent attachment of the Cy3 fluorophore was
shown with gel electrophoresis.
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12- alkyne-etheno-adenine modificationbioorthogonal groupcovalent attachmentpostsynthetic incorporationacid derivativesyields decreasestrandSequence-Specific Post-Synthetic OligonucleotideRNA moleculegel electrophoresisCy 3 fluorophore633 nucleotideDNA-templated synthesisSaccharomyces cerevisiae group II intron SclengthsmFRET measurementssingle-moleculestrategysolid-phase methodssequence-specific fluorescencefluorophore Cy 3Single-Molecule Fluorescence Applications
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