Structure, stability and elasticity of DNA nanotube

DNA nanotubes are tubular structures composed of DNA crossover molecules. We present a bottom up approach for construction and characterization of these structures. Various possible topologies of nanotubes are constructed such as 6-helix, 8-helix and tri-tubes with different sequences and lengths. We have used fully atomistic molecular dynamics simulations to study the structure, stability and elasticity of these structures. Several nanosecond long MD simulations give the microscopic details about DNA nanotubes. Based on the structural analysis of simulation data, we show that 6-helix nanotubes are stable and maintain their tubular structure; while 8-helix nanotubes are flattened to stabilize themselves. We also comment on the sequence dependence and effect of overhangs. These structures are approximately four times more rigid having stretch modulus of ~4000 pN compared to the stretch modulus of 1000 pN of DNA double helix molecule of same length and sequence. The stretch moduli of these nanotubes are also three times larger than those of PX/JX crossover DNA molecules which have stretch modulus in the range of 1500-2000 pN. The calculated persistence length is in the range of few microns which is close to the reported experimental results on certain class of the DNA nanotubes.Comment: Published in Physical Chemistry Chemical Physic

Single-Molecule Detection of Unique Genome Signatures: Applications in Molecular Diagnostics and Homeland Security

Single-molecule detection (SMD) offers an attractive approach for identifying the presence of certain markers that can be used for in vitro molecular diagnostics in a near real-time format. The ability to eliminate sample processing steps afforded by the ultra-high sensitivity associated with SMD yields an increased sampling pipeline. When SMD and microfluidics are used in conjunction with nucleic acid-based assays such as the ligase detection reaction coupled with single-pair fluorescent resonance energy transfer (LDR-spFRET), complete molecular profiling and screening of certain cancers, pathogenic bacteria, and other biomarkers becomes possible at remarkable speeds and sensitivities with high specificity. The merging of these technologies and techniques into two different novel instrument formats has been investigated. (1) The use of a charge-coupled device (CCD) in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement by simultaneously tracking and detecting single-molecules in multiple microfluidic channels was demonstrated. The CCD/TDI approach allowed increasing the sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s-1 per channel and 2208 molecules s-1 for an eight channel microfluidic system was achieved. A cyclic olefin copolymer (COC) waveguide was designed and fabricated in a pre-cast poly(dimethylsiloxane) stencil to increase the SNR by controlling the excitation geometry. The waveguide showed an attenuation of 0.67 dB/cm and the launch angle was optimized to increase the depth of penetration of the evanescent wave. (2) A compact SMD (cSMD) instrument was designed and built for the reporting of molecular signatures associated with bacteria. The optical waveguides were poised within the fluidic chip at orientation of 90° with respect to each other for the interrogation of single-molecule events. Molecular beacons (MB) were designed to probe bacteria for the classification of Gram +. MBs were mixed with bacterial cells and pumped though the cSMD which allowed S. aureus to be classified with 2,000 cells in 1 min. Finally, the integration of the LDR-spFRET assay on the cSMD was explored with the future direction of designing a molecular screening approach for stroke diagnostics

Polymer-Based Microfluidic Devices for High Throughput Single Molecule Detection: Applications in Biological and Drug Discovery

The realization of high throughput sample processing has become a primary ambition in many research applications with an example being high throughput screening (HTS), which represents the first step in the drug discovery pipeline. Microfluidics is a viable platform for parallel processing of biochemical reactions to increase data production rates due to its ability to generate fluidic networks with a high number of processors over small footprints suitable for optical imaging. Single-molecule detection (SMD) is another technology which has emerged to facilitate the realization of high throughput data processing afforded by its ability to eliminate sample processing steps and generate results with high statistical accuracy. A combination of microfluidics and SMD with wide-field fluorescence detection provides the ability to monitor biochemical reactions in a high throughput format with single-molecule sensitivity. In this dissertation, the integration of these techniques was presented for HTS applications in drug discovery. An ultra-sensitive fluorescence detection system with a wide field-of-view (FoV) was constructed to transduce fluorescence signatures from single chromophores that were electrokinetically transported through a series of tightly packed fluidic channels poised on poly(methylmethacrylate), PMMA and contained within the FoV of a laser detection system. The system was used to monitor biochemical reactions at the single-molecule level in a continuous-flow format. Enhancement in sampling-throughput was demonstrated by constructing a high density fluidic network for parallel analysis of multiple biochemical assays. In another development, the ability to enhance single-molecule sensitivity in a flow-based biochemical assay was investigated using a novel cyclic olefin copolymer (COC) planar waveguide embedded in PMMA and situated orthogonal to multiple fluidic channels. This design allowed for fluorescence detection from multiple fluidic channels using evanescent excitation and a wide FoV fluorescence detection system for parallel readout. Results from these technologies were presented as well as their applications in drug discovery for increasing data production rates and quality. An approach toward monitoring the efficacy of therapeutic agents, which is important in clinical evaluation of drug potency in the drug discovery process, was also considered, by designing a microfluidic system with integrated conductivity sensor for label-free enumeration of isolated tumor cells from clinical samples

Single-molecule detection and microfluidics: generating systems for the in vitro diagnostics of stroke

There is currently no available molecular diagnostic test for stroke; the common modality for diagnosis consists of computed tomography or magnetic resonance imaging. Unfortunately, the use of these diagnostic regimens can delay proper therapeutic treatment, which requires administration within the first 3 h of a stroke event. We are developing a molecular assay that can report, in near real time and at the point-of-care, the presence or absence of biomarkers specifically targeted for the diagnosis of ischemic or hemorrhagic stroke. The proposed strategy uses blood-borne mRNAs that are either under-expressed or over-expressed as a result of tissue damage within the brain. The ability to report on these diagnostic markers is enabled through the use of a fluidic bio-processor fabricated in polymers via micro-replication to provide autonomous sample processing. This bio-processor comprises a fluidic motherboard that possesses task-specific modules for the selection of white blood cells from a blood sample, cell lysis and solid-phase extraction of the mRNA markers, ligase detection reaction to identify the mRNA markers and an optical module for multiplexed detection. The sample-processing pipeline was streamlined to generate a rapid assay turn-around-time by employing single-molecule detection. The output of the clinical sample processing hardware are molecular beacons undergoing single pair Fluorescence Resonance Energy Transfer (spFRET) that are digitally counted to provide exquisite analytical sensitivity for the expression profiling of the relevant mRNA markers. The presentation will discuss the use of spFRET for mRNA expression profile with comparisons made to quantitative real-time PCR

Cross-talk-free dual-color fluorescence cross-correlation spectroscopy for high-throughtput screening

High throughput processing of chemical/biochemical information is critical in many areas, such as genome sequencing, drug discovery and clinical diagnostics. Integral to collecting information at high rates with the necessary throughput is the development of systems that can not only monitor the results with high precision and accuracy, but also prepare and process samples prior to the analytical measurement. To achieve the required throughput, we have conducted work directed toward developing a system that provides detection sensitivity at the single-molecule detection (SMD) level. The research was first focused on the development of a sensitive strategy for the detection of proteins (thrombin) at the SMD level. Nucleic acid-based fluorescence sensors were used as recognition elements for the detection of single protein molecules with single-pair fluorescence resonance energy transfer. The technique provided higher analytical sensitivity compared to bulk analog measurements due to the digital readout format (i.e., molecular counting) and also reduced assay turn-around-time. Research was then directed toward the design and construction of a two-color FCCS system, which employed two spectrally separate fluorophores, Cy3 (λabs = 532 nm, λem= 560 nm) and IRD800 (λabs = 780 nm, λem= 810 nm). The system provided negligible color cross-talk (cross-excitation and/or cross-emission) and/or fluorescence resonance energy transfer (FRET). To provide evidence of cross-talk free operation, the system was evaluated using microspheres and quantum dots. Experimental results indicated no color leakage from the microspheres or quantum dots into inappropriate color channels. The enzymatic activity of APE1 was monitored by FCCS using a double-stranded DNA substrate that was dual labeled with Cy3 and IRD800. Activity of APE1 was also monitored in the presence of an inhibitor (7-nitroindole-2-carboxylic acid). To improve sample processing throughput, a multi-phase (water-in-oil) segmented flow microfluidic chip was studied using the FCCS system to monitor APE1 enzyme activity. Aqueous droplets were generated in a perfluorocarbon (FC-3283) carrier fluid with a nonionic surfactant (Perfluorooctanol, 10% v/v) in a polymer microchip. The optical system successfully monitored the controlled generation of highly regular droplets loaded with fluorescent beads at delivery rates ranging from 40 - 60 droplets per sec

VisANT 3.0: new modules for pathway visualization, editing, prediction and construction

With the integration of the KEGG and Predictome databases as well as two search engines for coexpressed genes/proteins using data sets obtained from the Stanford Microarray Database (SMD) and Gene Expression Omnibus (GEO) database, VisANT 3.0 supports exploratory pathway analysis, which includes multi-scale visualization of multiple pathways, editing and annotating pathways using a KEGG compatible visual notation and visualization of expression data in the context of pathways. Expression levels are represented either by color intensity or by nodes with an embedded expression profile. Multiple experiments can be navigated or animated. Known KEGG pathways can be enriched by querying either coexpressed components of known pathway members or proteins with known physical interactions. Predicted pathways for genes/proteins with unknown functions can be inferred from coexpression or physical interaction data. Pathways produced in VisANT can be saved as computer-readable XML format (VisML), graphic images or high-resolution Scalable Vector Graphics (SVG). Pathways in the format of VisML can be securely shared within an interested group or published online using a simple Web link. VisANT is freely available at http://visant.bu.edu

VisANT 3.0: new modules for pathway visualization, editing, prediction and construction

With the integration of the KEGG and Predictome databases as well as two search engines for coexpressed genes/proteins using data sets obtained from the Stanford Microarray Database (SMD) and Gene Expression Omnibus (GEO) database, VisANT 3.0 supports exploratory pathway analysis, which includes multi-scale visualization of multiple pathways, editing and annotating pathways using a KEGG compatible visual notation and visualization of expression data in the context of pathways. Expression levels are represented either by color intensity or by nodes with an embedded expression profile. Multiple experiments can be navigated or animated. Known KEGG pathways can be enriched by querying either coexpressed components of known pathway members or proteins with known physical interactions. Predicted pathways for genes/proteins with unknown functions can be inferred from coexpression or physical interaction data. Pathways produced in VisANT can be saved as computer-readable XML format (VisML), graphic images or high-resolution Scalable Vector Graphics (SVG). Pathways in the format of VisML can be securely shared within an interested group or published online using a simple Web link. VisANT is freely available at http://visant.bu.edu

Single-molecule detection of molecular beacons generated from LDR on thermoplastic microfluidic device for bioanalysis

Current clinical techniques for nucleic acid detection and analysis often involve PCR, lacking adequate specificity or sensitivity to meet the stringent requirements in certain applications. This research aims to develop an innovative molecular assay and the associated hardware to rapidly signal the presence of certain targets using reporter sequence found in their genome without requiring PCR. This assay coupled the sensitivity of single-pair fluorescence resonance energy transfer (spFRET) with the specificity of ligase detection reaction (LDR) to provide near real-time readout of target biomarkers. Heightened concerns on potential bioterrorism threats, such as rapid dissemination of pathogenic bacteria or viruses into water and/or food supplies, demand fast detection strategies. In this work, a pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) and acceptor (Cy5.5) dyes. In the presence of the target bacterium, the primers were joined using LDR to form a reverse molecular beacon (rMB), thus bringing Cy5 and Cy5.5 into close proximity to allow FRET to occur. These rMBs were analyzed using single-molecule detection of the FRET pairs (spFRET). The LDR was performed in a Cyclic Olefin Copolymer (COC) microfluidic device equipped with 2 or 20 thermal cycles in a continuous flow format. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a laser-induced fluorescence (LIF) instrument. The presence of target pathogens could be reported in as little as 2.6 min using spFRET. In another development, a similar assay format was utilized to quantify mRNA expression levels of MMP-7 gene, which is highly relevant to invasion, metastasis and progression of a variety of tumors. HT-29 cells were found to express the highest levels of MMP-7 transcripts among the studied cell lines using LDR primers specific to MMP-7 gene. This observation is consistent with the results obtained with RT-qPCR. The LDR-spFRET assay was also used for stroke subtyping by designing primers specific to AMPH gene and using a microfluidic chip with tapered detection window to improve sampling efficiency. The detection could be completed in 15 min with extended readout time to glean low copy number transcripts