(141a) Thermal Fluctuations of Single DNA Molecules in Nanoconfinement

In this talk, we report the first experimental proof of weak excluded volume effects when DNA is confined to a nanochannel in the extended de Gennes regime. This result is important to the fundamental understanding of confined semiflexible chains like DNA, providing experimental confirmation of a key theoretical prediction [1-3].

Manipulation of single DNA molecules in nanochannels has emerged as an important platform for genome mapping. Originally, the confinement of a semiflexible polymer in a pore was described as a self-avoiding walk of Odijk deflection segments (for strong confinement) or isometric de Gennes blobs (for weak confinement). It is now understood that there is a rich transition behavior for a semi-flexible polymer like DNA [3]. In the extended de Gennes regime, which governs channel sizes l_p < D < l_p²/w (for a polymer with persistence length l_p and effective width w), the weak self-exclusion of the chain at the local level makes the chain swell in the axial direction. The result of this weak excluded volume effects is self-avoiding, anisometric blobs at the global scale. To experimentally differentiate the anisometric blobs associated with the extended de Gennes regime from their isometric counterparts in the de Gennes regime, which have strong excluded volume, the variance in extension at equilibrium becomes the relevant parameter because it acts as a probe of the local blob statistics [1].

Our device consists of two 1-μm deep and 50-μm wide microchannels, bridged by an array of nanofunnels. A single nanofunnel has ten connected 300 nm deep and 45-μm long nanochannels; the width of the nanochannel varies from 300 nm to 750 nm in steps of 50 nm. It is similar in design to our previous 100 nm deep, equilibrium confinement spectroscopy device [4]. Fabrication of the device was done on a fused silica wafer using a layer of electron beam lithography for nanofunnels and a layer of UV contact lithography for microchannels, each followed by a reactive ion etching step. Next, a T4 phage DNA molecule labeled with YOYO-1 dye molecules was introduced inside the nanofunnel using pneumatic pressure [4], equilibrated for 60s at a desired channel size, and imaged for 40 frames at an interval of at least 5s between frames (maximum relaxation time ≈ 2.5s). We tracked each DNA molecule in all the channel sizes to reduce the error arising from the distribution of sizes in a DNA sample. Extension measurements from 29 molecules were used to calculate the average extension as a function of the channel size. The variance in extension for each molecule was then pooled together to calculate the average fluctuation in extension.

The average extension has a power law dependence on the channel size with exponent = -0.77±0.05, which is stronger than the prediction for the extended de Gennes regime (-0.67) [2] but weaker than the previous experiments (-0.85) for relatively smaller channels [4]. In contrast, the average fluctuation in extension is independent of the channel size, as predicted by theory for the extended de Gennes regime [1]. Moreover, the numerical value agrees with the exact asymptotic solution [2]. We conclude that the weak excluded volume interactions, characteristic of the extended de Gennes regime, are present for channel sizes much greater than the persistence length of the chain.

REFERENCES:

[1]      L. Dai, J. van der Maarel, and P. S. Doyle, Macromolecules 47, 2445 (2014).

[2]      E. Werner and B. Mehlig, Phys. Rev. E 90, 062602 (2014).

[3]      T. Odijk, Phys. Rev. E 77, 060901(R) (2008).

[4]       D. Gupta, J. Sheats, A. Muralidhar, J. J. Miller, D. E. Huang, S. Mahshid, K. D. Dorfman, and W. Reisner, J. Chem. Phys. 140, 214901  (2014).