Benjamin R. Cipriany

Single Molecule Epigenetic Analysis in a Nanofluidic Channel

Epigenetic states are governed by DNA methylation and a host of modifications to histones bound with DNA. These states are essential for proper developmentally regulated gene expression and are perturbed in many diseases. There is great interest in identifying epigenetic mark placement genome wide and understanding how these marks vary among cell types, with changes in environment or according to health and disease status. Current epigenomic analyses employ bisulfite sequencing and chromatin immunoprecipitation, but query only one type of epigenetic mark at a time, DNA methylation, or histone modifications and often require substantial input material. To overcome these limitations, we established a method using nanofluidics and multicolor fluorescence microscopy to detect DNA and histones in individual chromatin fragments at about 10 Mbp/min. We demonstrated its utility for epigenetic analysis by identifying DNA methylation on individual molecules. This technique will provide the unprecedented opportunity for genome wide, simultaneous analysis of multiple epigenetic states on single molecules.

Fabrication of a Nanomechanical Mass Sensor Containing a Nanofluidic Channel

Nanomechanical resonators operating in vacuum are capable of detecting and weighing single biomolecules, but their application to the life sciences has been limited by viscous forces that impede their motion in liquid environments. A promising approach to avoid this problem, encapsulating the fluid within a mechanical resonator surrounded by vacuum, has not yet been tried with resonant sensors of mass less than approximately 100 ng, despite predictions that devices with smaller effective mass will have proportionally finer mass resolution. Here, we fabricate and evaluate the performance of doubly clamped beam resonators that contain filled nanofluidic channels and have masses of less than 100 pg. These nanochannel resonators operate at frequencies on the order of 25 MHz and when filled with fluid have quality factors as high as 800, 2 orders of magnitude higher than that of resonators of comparable size and frequency operating in fluid. Fluid density measurements reveal a mass responsivity of 100 Hz/fg and a noise equivalent mass of 2 fg. Our analysis suggests that realistic improvements in the quality factor and frequency stability of nanochannel resonators would render these devices capable of sensing attogram masses from liquid.

Photothermal Self-Oscillation and Laser Cooling of Graphene Optomechanical Systems

By virtue of their low mass and stiffness, atomically thin mechanical resonators are attractive candidates for use in optomechanics. Here, we demonstrate photothermal back-action in a graphene mechanical resonator comprising one end of a Fabry-Perot cavity. As a demonstration of the utility of this effect, we show that a continuous wave laser can be used to cool a graphene vibrational mode or to power a graphene-based tunable frequency oscillator. Owing to graphenes high thermal conductivity and optical absorption, photothermal optomechanics is efficient in graphene and could ultimately enable laser cooling to the quantum ground state or applications such as photonic signal processing.

Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells

Proper placement of epigenetic marks on DNA and histones is fundamental to normal development, and perturbations contribute to a variety of disease states. Combinations of marks act together to control gene expression; therefore, detecting their colocalization is important, but because of technical challenges, such measurements are rarely reported. Instead, measurements of epigenetic marks are typically performed one at a time in a population of cells, and their colocalization is inferred by association. Here, we describe a single-molecule analytical approach that can perform direct detection of multiple epigenetic marks simultaneously and use it to identify mechanisms coordinating placement of three gene silencing marks, trimethylated histone H3 lysine 9, lysine 27 (H3K9me3, H3K27me3), and cytosine methylation (mC), in the normal and cancer genome. We show that H3K9me3 and mC are present together on individual chromatin fragments in mouse embryonic stem cells and that half of the H3K9me3 marks require mC for their placement. In contrast, mC and H3K27me3 coincidence is rare, and in fact, mC antagonizes H3K27me3 in both embryonic stem cells and primary mouse fibroblasts, indicating this antagonism is shared among primary cells. However, upon immortalization or tumorigenic transformation of mouse fibroblasts, mC is required for complete H3K27me3 placement. Importantly, in human promyelocytic cells, H3K27me3 is also dependent on mC. Because aberrant placement of gene silencing marks at tumor suppressor genes contributes to tumor progression, the improper dependency of H3K27me3 by mC in immortalized cells is likely to be fundamental to cancer. Our platform can enable other studies involving coordination of epigenetic marks and leverage efforts to discover disease biomarkers and epigenome-modifying drugs.

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.

Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells

We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

Single DNA molecule patterning for high-throughput epigenetic mapping

We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays estimated to contain more than 250 000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are fixed in their extended configuration by adsorption onto the substrate so analysis can be performed with high spatial resolution and signal averaging. We further prove this technique allows imaging of DNA molecules with different methylation states.

Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

Laser induced fluorescence in submicrometer fluidic channels was used to characterize the synthesis of polymerase chain reaction (PCR) products from a model bacterial system in order to explore the advantages and limitations of on chip real time single molecule PCR analysis. Single oligonucleotide universal bacterial primers and PCR amplicons from the 16S rDNA of Thermobifida fusca (325 bp) were directly detected at all phases of the reaction with low sample consumption and without post-amplification purification or size screening. Primers were fluorescently labeled with single Alexa Fluor 488 or Alexa Fluor 594 fluorophores, resulting in double labeled, two color amplicons. PCR products were driven electrokinetically through a fused silica channel with a 250 nm by 500 nm rectangular cross section. Lasers with 488 nm and 568 nm wavelengths were focused and overlapped on the channel for fluorescence excitation. All molecules entering the channel were rapidly and uniformly analyzed. Photon burst analysis was used to detect and identify individual primers and amplicons, and fluorescence correlation and cross-correlation spectroscopy were used to account for analyte flow speed. Conventional gel and capillary electrophoresis were also used to characterize the PCR amplification, and the results of differences in detection sensitivity and analyte discrimination were examined. Limits were imposed by the purity and labeling efficiency of the PCR reagents, which must be improved in parallel with increases in detection sensitivity.

Labeling and purification of cellulose-binding proteins for high resolution fluorescence applications

The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores. To have meaningful interpretations of results from the study of labeled enzymes, it is then necessary to reduce the variability in physical, chemical, and biological characteristics of the labeled products. In this paper, a solid phase labeling protocol is described as an advantageous alternative to free solution labeling of cellulose-binding proteins and is applied to tag cellulases with three different fluorophores. The products from the labeling reactions were purified to remove the unreacted dye and separate labeled and unlabeled enzymes. Characterization of the catalytic and spectroscopic properties of the isolated labeled species confirmed that highly homogeneous populations of labeled cellulases can be achieved. The protocol for the separation of labeled products is applicable to any mixture of labeled proteins, making this an attractive methodology for the production of labeled proteins suitable for single molecule fluorescence spectroscopy.

Real-time synchronous imaging of electromechanical resonator mode and equilibrium profiles.

The single-shot interferometric imaging of normal mode dynamics in MEMS resonators, oscillating in the radio frequency (rf) regime, is demonstrated by synchronous imaging with a pulsed nanosecond laser. Profiles of mechanical modes in suspended silicon and graphene thin-film structures, and their extracted equilibrium profiles, are measured through nondestructive all-optical Fabry-Perot reflectance fits to the temporal traces. As a proof of principle, the modal patterns of a microdrum silicon resonator is analyzed, and its extracted vibration modes and equilibrium profile show good agreement with other characterization and numerical estimations.