Walter Reisner

DNA confinement in nanochannels: physics and biological applications

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement--including the effect of varying ionic strength--and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Single-molecule denaturation mapping of DNA in nanofluidic channels

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent “barcode” corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.

Confinement Spectroscopy: Probing Single DNA Molecules with Tapered Nanochannels

We demonstrate a confinement spectroscopy technique capable of probing small conformational changes of unanchored single DNA molecules in a manner analogous to force spectroscopy, in the regime corresponding to femtonewton forces. In contrast to force spectroscopy, various structural forms of DNA can easily be probed, as indicated by experiments on linear and circular DNA. The extension of circular DNA is found to scale according to the de Gennes exponent, unlike for linear DNA.

Thermocapillary actuation of liquid flow on chemically patterned surfaces

We have investigated the thermocapillary flow of a Newtonian liquid on hydrophilic microstripes which are lithographically defined on a hydrophobic surface. The speed of the microstreams is studied as a function of the stripe width w, the applied thermal gradient |dT/dx| and the liquid volume V deposited on a connecting reservoir pad. Numerical solutions of the flow speed as a function of downstream position show excellent agreement with experiment. The only adjustable parameter is the inlet film height, which is controlled by the ratio of the reservoir pressure to the shear stress applied to the liquid stream. In the limiting cases where this ratio is either much smaller or much larger than unity, the rivulet speed shows a power law dependency on w, |dT/dx| and V. In this study we demonstrate that thermocapillary driven flow on chemically patterned surfaces can provide an elegant and tunable method for the transport of ultrasmall liquid volumes in emerging microfluidic technologies.

Directed self-organization of single DNA molecules in a nanoslit via embedded nanopit arrays

We show that arrays of nanopit structures etched in a nanoslit can control the positioning and conformation of single DNA molecules in nanofluidic devices. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of a device without additional chemical modification and achieve a high degree of control over local DNA conformation. DNA can be extended between two nanopits and in closely spaced arrays will self-assemble into “connect-the-dots” conformations consisting of locally pinned segments joined by fluctuating linkers. These results have broad implications for nanotechnology fields that require methods for the nanoscale positioning and manipulation of DNA.

Convex lens-induced nanoscale templating

Significance Convex lens-induced nanoscale templating (CLINT) represents a conceptual breakthrough in nanofluidic technology for single-molecule manipulation. CLINT solves a key challenge faced by the nanofluidics field by bridging the multiple-length scales required to efficiently bring single-molecule analytes from the pipette tip to the nanofluidic channel. To do this, CLINT loads single-molecule analytes into embedded nanofeatures via dynamic control of applied vertical confinement, which we have demonstrated by loading and extending DNA within nanochannels. CLINT offers unique advantages in single-molecule DNA mapping by facilitating surface passivation, increasing loading efficiency, obviating the need for applied pressure or electric fields, and enhancing compatibility with physiological buffers and long DNA molecules extracted from complex genomes. We demonstrate a new platform, convex lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a convex lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub–30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.

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.

Fabrication and characterization of nanopore-interfaced nanochannel devices

Nanofluidic devices combining nanochannels and nanopores may enable a range of novel applications in the field of single-molecule biosensing and manipulation. Here we combine classic lithographically based fabrication and electron beam milling to construct a device that integrates sealed transverse features, such as nanocavities and nanochannels, with embedded pores vertically intersecting the nanochannels. Using fluorescent microscopy, we demonstrate that DNA molecules can be introduced into the nanochannels and translated transversely across the embedded pore in an extended-conformation without undergoing cross-pore translocation. Upon application of a trans-pore voltage drop, the molecules will undergo cross-pore translocation into an adjoining macroscopic reservoir.

Transition state theory demonstrated at the micron scale with out-of-equilibrium transport in a confined environment

Transition state theory (TST) provides a simple interpretation of many thermally activated processes. It applies successfully on timescales and length scales that differ several orders of magnitude: to chemical reactions, breaking of chemical bonds, unfolding of proteins and RNA structures and polymers crossing entropic barriers. Here we apply TST to out-of-equilibrium transport through confined environments: the thermally activated translocation of single DNA molecules over an entropic barrier helped by an external force field. Reaction pathways are effectively one dimensional and so long that they are observable in a microscope. Reaction rates are so slow that transitions are recorded on video. We find sharp transition states that are independent of the applied force, similar to chemical bond rupture, as well as transition states that change location on the reaction pathway with the strength of the applied force. The states of equilibrium and transition are separated by micrometres as compared with angstroms/nanometres for chemical bonds.

Transverse dielectrophoretic-based DNA nanoscale confinement

Confinement of single molecules within nanoscale environments is crucial in a range of fields, including biomedicine, genomics, and biophysics. Here, we present a method that can concentrate, confine, and linearly stretch DNA molecules within a single optical field of view using dielectrophoretic (DEP) force. The method can convert an open surface into one confining DNA molecules without a requirement for bonding, hydrodynamic or mechanical components. We use a transverse DEP field between a top coverslip and a bottom substrate, both of which are coated with a transparent conductive material. Both layers are attached using double-sided tape, defining the chamber. The nanofeatures lie at the “floor” and do not require any bonding. With the application of an alternating (AC) electric field (2 Vp-p) between the top and bottom electrodes, a DEP field gradient is established and used to concentrate, confine and linearly extend DNA in nanogrooves as small as 100-nm in width. We also demonstrate reversible loading/unloading of DNA molecules into nanogrooves and nanopits by switching frequency (between 10 kHz to 100 kHz). The technology presented in this paper provides a new method for single-molecule trapping and analysis.