Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro-and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.

Fully stretched DNA molecules are becoming a fundamental component of new systems for comprehensive genome analysis. Among a number of approaches for elongating DNA molecules, nanofluidic molecular confinement has received enormous attentions from physical and biological communities for the last several years. Here we demonstrate a well-optimized condition that a DNA molecule can stretch almost its full contour length: the average stretch is 19.1 μm ± 1.1 μm for YOYO-1 stained λ DNA (21.8 μm contour length) in 250 nm × 400 nm channel, which is the longest stretch value ever reported in any nanochannels or nanoslits. In addition, based on Odijk’s polymer physics theory, we interpret our experimental findings as a function of channel dimensions and ionic strengths. Furthermore, we develop a Monte Carlo simulation approach using a primitive model for the rigorous understandings of DNA confinement effects. Collectively, we present more complete understanding of nanochannel confined DNA stretching via the comparisons to computer simulation results and Odijk’s polymer physics theory.

RNA polymerase (RNAP) is one of the best characterized motor proteins which transcribes on a DNA template while presenting unique motor properties. 1 The powerful tracking ability of RNAP underlies devices supporting nanoscale motion control on planar substrates. 2 Nevertheless existing experimental systems that track protein motor activities are restricted to tethering the substrate or the motor, therefore cannot support observation of autonomous movement modulated by the motor in free solution. Although autonomous control has been reported by metallic, reactive colloids, 3 it has not been demonstrated with biomolecules under physiological conditions. Here we show the first experimental evidence that a molecular complex consisting of just a DNA template and associated RNAPs displays chemokinetic motion driven by transcription substrate NTPs. Furthermore this molecular complex exhibits biased migration into a concentration gradient of NTPs for mimicking cellular chemotaxis.We studied a robust transcription system using T7 RNAP and a rod-like 310 bp DNA template bearing a T7 transcription promoter sequence. For detection, the upstream portion from the promoter of the template was fluorescently labeled. Apparent diffusion of DNA was quantified by Fluorescence Recovery After Photobleaching (FRAP). 4 The diffusion coefficient (D) for the 310 bp DNA in transcription buffer without Mg 2+ was 15.3 μm 2 /s, which is 18.6% slower than the 18.8 μm 2 /s value obtained from theoretical calculations via Broersma's equations. 5 We attribute this difference to impedance of DNA motion by the gel matrix (added to eliminate convection) and fluorochrome labels attached to the template.We evaluated motility of the DNA-RNAP complex in the presence of substrate NTPs as compared to controls prepared from same aliquots of reagents, but without NTPs substrates ( Figure 1). We observed a systematic increase (25.2% on average) of apparent diffusion of DNA when transcription is enabled. Our control showed that DNA diffusion is invariant over the ionic strength range examined in our experiments. Increase in ionic strength by monovalent Li + (included in added NTPs) should not significantly affect diffusivity. Therefore, RNAP motor activity must be the major cause that renders faster movement of DNA -a phenomenon we describe as "Molecular Propulsion." While transcribing on the DNA track, the RNAP motor vigorously pushes and pulls on the template DNA against fluid viscous drag, affecting hydrodynamic interactions between DNA and surrounding water layers. We further examined transcription elongation and initiation as possible governing factors in Molecular Propulsion (Figure 2). By adding 3'-dNTPs to the NTPs at 1:100 ratio, the length of transcription elongation is truncated from the runoff full length (227 nt) to roughly 100 nt.Comparisons of apparent DNA diffusion between runoff transcription and 100 nt transcription showed no significant difference beyond experimental fluctuation. Increasing the ratio of 3'-dNTP vs. NTP to 1:10 traps the m...

The conformation and dynamics of a single DNA molecule undergoing oscillatory pressure-driven flow in microfluidic channels is studied using Brownian dynamics simulations, accounting for hydrodynamic interactions between segments in the bulk and between the chain and the walls. Oscillatory flow provides a scenario under which the polymers may remain in the channel for an indefinite amount of time as they are stretched and migrate away from the channel walls. We show that by controlling the chain length, flow rate and oscillatory flow frequency, we are able to manipulate the chain extension and the chain migration from the channel walls. The chain stretch and the chain depletion layer thickness near the wall are found to increase as the Weissenberg number increases and as the oscillatory frequency decreases.

Much of modern biology relies on the strategic manipulation of molecules for creating ordered arrays prior to high throughput molecular analysis. Normally, DNA arrays involve deposition on surfaces, or confinement in nanochannels; however, we show that microfluidic devices can present stretched molecules within a controlled flow in ways complementing surface modalities, or extreme confinement conditions. Here we utilize pressure-driven oscillatory shear flows generated in microchannels as a new way of stretching DNA molecules for imaging “arrays” of individual DNA molecules. Fluid shear effects both stretch DNA molecules and cause them to migrate away from the walls becoming focused in the centerline of a channel. We show experimental findings confirming simulations using Brownian dynamics accounting for hydrodynamic interactions between molecules and channel-flow boundary conditions. Our findings characterize DNA elongation and migration phenomena as a function of molecular size, shear rate, oscillatory frequency with comparisons to computer simulation studies.