Paper BI+AS+NS-MoA11
Measuring DNA Looping Pathways using Nanofluidic Manipulation

Monday, November 10, 2014, 5:20 pm, Room 317

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.

In this paper we will discuss a group of nanofluidic device that force two DNA molecules to either slide past each other in parallel, or cross over each other at a steep angle. Nanochannel cross-sections are 100x100 nm², and are hundreds of microns long. Because DNA is elongated through confinement, loop 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 will present analyses of different DNA-binding proteins that demonstrate that we can distinguish dense and sparse binding modes and the compensation of electrostatic DNA-DNA repulsion through protein binding. We further report the detection of long-lived tertiary complexes acting as a lock for looped DNA configurations, and the presence of very short-lived transient links. We further demonstrate a pathway for loop formation that is enhanced in nanochannel devices, and that may be important in a cellular context. By using precision hydrodynamic flows, we are able to measure free energies of the search process.