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Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging.

ABSTRACT: Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.

SUBMITTER: Bell NAW

PROVIDER: S-EPMC9303394 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging.

Bell Nicholas A W NAW Molloy Justin E JE

Nucleic acids research 20220701 13

Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate a ...[more]

PMID: 35489063

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