Electrophoretic stretching and imaging of single native chromatin fibers in nanoslits.

Stretching single chromosomal DNA fibers in nanofluidic devices has become a valuable tool for studying the genome and more recently the epigenome. Although nanofluidic technology has been extensively used in single molecular DNA analysis, compared to bare DNA, much less work has been done to elongate chromatin, and only a few studies utilize more biologically relevant samples such as native eukaryotic chromatin. Here, we provide a method for stretching and imaging individual chromatin fibers within a micro- and nanofluidic device. This device was used to electrophoretically stretch and image single native chromatin fibers extracted from human cancer cells (HeLa cells) by attaching the chromatin to microspheres held at the entrance of a nanoslit. To further demonstrate the potential of this device in epigenetics, histone modification H3k79me2 was optically detected by fluorescence microscopy.

Stretching of Tethered DNA in Nanoslits.

External forces and confinement are two fundamental and complementary approaches for biopolymer stretching. By employing micro- and nanofluidics, we study the force-extension dynamics by simultaneously applying external forces and confinement to single-DNA molecules. In particular, we apply external electric fields to stretch single DNA molecules that are attached to microspheres anchored at a nanoslit entrance. Using this method, we measure the force-extension relation of tethered DNA and describe this relation with modified wormlike chain models. This allowed experimental validations of several theoretical predictions, including the increase in the global persistence length of confined DNA with increasing degree of confinement and the "confined Pincus" regime in slit confinement.

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices.

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (∼ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.

Entropy-driven single molecule tug-of-war of DNA at micro-nanofluidic interfaces.

Entropy-driven polymer dynamics at the nanoscale is fundamentally important in biological systems but the dependence of the entropic force on the nanoconfinement remains elusive. Here, we established an entropy-driven single molecule tug-of-war (TOW) at two micro-nanofluidic interfaces bridged by a nanoslit, performed the force analysis from a modified wormlike chain in the TOW scenario and the entropic recoiling process, and determined the associated scalings on the nanoconfinement. Our results provide a direct experimental evidence that the entropic forces in these two regimes, though unequal, are essentially constant at defined slit heights, irrespective of the slit lengths and the DNA segments within. Our findings have the implications to polymer transport at the nanoscale, device design for single molecule analysis, and biotechnological applications.