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Sequence-specific post-synthetic oligonucleotide labeling for single-molecule fluorescence applications


Egloff, David; Oleinich, Igor A; Zhao, Meng; König, Sebastian L B; Sigel, Roland K O; Freisinger, Eva (2016). Sequence-specific post-synthetic oligonucleotide labeling for single-molecule fluorescence applications. ACS Chemical Biology, 11(9):2558-2567.

Abstract

The 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.

Abstract

The 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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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2016
Deposited On:24 Oct 2016 14:45
Last Modified:29 May 2017 07:25
Publisher:American Chemical Society (ACS)
ISSN:1554-8929
Publisher DOI:https://doi.org/10.1021/acschembio.6b00343
PubMed ID:27409145

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