Yan Mei Wang

Nanochannels for Genomic DNA Analysis: The Long and the Short of It

This review will discuss the theory of confined polymers in nanochannels and present our experiments, which test the theory and explore use of nanochannels for genomic analysis. Genomic length DNA molecules contained in nanochannels, which are less than one persistence length in diameter, are highly elongated. Thus, nanochannels can be used to analyze genomic length DNA molecules with very high linear spatial resolution. Also, nanochannels can be used to study the position and dynamics of proteins such as transcription factors that bind to DNA with high specificity. In order to realize these goals not only must nanochannels be constructed whose radius is less than the persistence length of DNA, but it is also necessary to understand the dynamics of polymers within nanochannels and develop experimental tools to study the dynamics of polymers in such confined volumes, tools which we review here.

Long-range interactions between transcription factors.

We discuss a method for analysing the number of GFP-LacI fusion transcription factors bound to a construct of 256 contiguous LacI binding sites using photon bleaching statics. We show by using a combination of imaging of the construct in nanochannels, photon statistics and addition of IGFP that the binding coefficient of the LacI decreases with increasing occupation of the construct, with a binding coefficient of 10(-6) M when only 15 of the 256 possible sites are occupied. We model this effect by assuming that the GFP-LacI dimer introduces elastic strain into the helix by generalized deformations, and that this strain propagates over distances at least as large as the persistence length.

When is a Distribution Not a Distribution, and Why Would You Care: Single-Molecule Measurements of Repressor Protein 1-D Diffusion on DNA

We address the long-standing puzzle of why some proteins find their targets faster than allowed by 3D diffusion. To this end, we measured the one- dimensional diffusion of LacI repressor proteins along elongated Lambda DNA using single molecule imaging techniques. We find that (1) LacI diffuses along nonspecific sequences of DNA in the form of 1D Brownian motion; (2) the observed 1D diffusion coefficients DDNA vary over an unexpectedly large range, from 2.3 × 10 −12 cm 2 /s to 1.3 × 10 −9 cm 2 /s; (3) the lengths of DNA covered by these 1D diffusions vary from 120 nm to 2920 nm; and (4) the mean values of DDNA and the diffusional lengths indeed predict a LacI target binding rate 90 times faster than the 3D diffusion limit. The first half of this chapter is a tutorial on the models we use to think about the physics, the limited and noisy data, and how to squeeze the maximum amount of physics from these data. The second half is about our experiments and results.

Sequence Matters: The Influence of Basepair Sequence on DNA-protein Interactions

The sequencing of the human genome, along with the 200-odd other genomes that have been sequenced, does not represent the solution to a puzzle but rather the necessary introduction to a bigger puzzle. That puzzle is how all the 30,000-odd some genes in the human genome are expressed and controlled in a proper sequence for a cell to function. We can hardly address this enormous problem in this brief review, but instead wish to concentrate on one very small but very important aspect of this problem: physical aspects to how proteins are able to achieve base-pair specific recognition. By “physical aspects” we mean that the proteins distort (strain) the DNA helix when they bind, and if this strain is a function of the sequence of the distorted region then the basepair dependent free energy associated with the strain provides a way to discriminate amongst the basepairs.

DNA statics and dynamics in nanoscale confinement

We present, along with theoretical scaling arguments, measurements of the equilibrium and dynamic properties of λ and T2 phage DNA molecules confined in quartz nanochannels. Such measurements serve a two-fold purpose: (1) we hope to assist in the design of future nanofluidic devices by quantifying the behavior of semiflexible polymers in confined environments and (2) we hope to test existing theories for confined semiflexible polymers.