Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session II
40 – POS
Board 12
Detecting DNA Knotting by Strong Confinement and Action of Loop-inducing Proteins
Maedeh Roushan, Zubair Azad, Parminder Kaur, Jianguo Lin, Hong Wang,
Robert Riehn
.
North Carolina State University, Raleigh, NC, USA.
DNA performs a carefully choreographed ballet during the cell cycle. The organization is driven
by the specific binding of proteins to form tertiary DNA-protein-DNA complexes. The search
process that precedes the formation must overcome the challenge of very low effective mobility
of genomic-sized DNA pieces in the dense cellular environment, and potentially leads to
complex topologies such as knots and loops.
We have developed nanofluidic devices that gently elongate single DNA molecules through a
lateral force without tethering the molecule or blocking the molecule ends. Nanochannel cross-
sections are 100x100 nm2, and channels are hundreds of microns long. Because DNA is
elongated through confinement, loops and knots with a length down to 2 kb can be directly
observed in real time. Channels are made of fused silica, enabling single-molecule observation of
both DNA and proteins. Because the effective concentration of DNA inside channels exceeds 1
mg/ml with the channel at the point of DNA-DNA contact, protein-mediated capture cross-
sections are very high.
We have investigated a range of proteins that have been proposed as part of the telomeric T-loop
complex, as well as DNA ligases. We find that a subset of these induce knots that are free to
travel along the elongated DNA molecule, but cannot leave the DNA strand at its end. We
further find that we can distinguish these knots from simple loops, and that the diffusivity of
knots and loops can be modified through transient protein bridges. Finally we are able to
distinguish proteins with dense and sparse binding patterns.
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