Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

Langmuir. 2018 Dec 11;34(49):14882-14890. doi: 10.1021/acs.langmuir.8b01812. Epub 2018 Aug 2.


Single-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. Because ssDNA is more flexible than double-stranded (ds) DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate that low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription, and repair.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacillus Phages / enzymology
  • Base Sequence
  • DNA, Single-Stranded / chemical synthesis
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed DNA Polymerase / chemistry
  • Escherichia coli / chemistry
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism*
  • Fluorescence
  • Green Fluorescent Proteins / chemistry
  • Humans
  • Nucleic Acid Conformation / drug effects
  • Protein Binding
  • Protein Conformation
  • Replication Protein A / chemistry
  • Replication Protein A / metabolism*
  • Sodium Chloride / chemistry


  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Escherichia coli Proteins
  • RPA1 protein, human
  • Replication Protein A
  • SSB protein, E coli
  • Green Fluorescent Proteins
  • Sodium Chloride
  • DNA-Directed DNA Polymerase