Long Strands Of DNA Research Articles

8414 Utilization of Cell Free DNA Integrity Index to Differentiate Benign from Malignant Thyroid Nodules

Abstract Disclosure: S. Tarafdar: None. S. Dutta: None. P. Mukhopadhyay: None. N.P. Bhattacharya: None. S. Ghosh: None. Introduction: Recent advances in molecular genetics have evaluated a potential role for plasma cell-free DNA (cfDNA) integrity index as a non-invasive cancer marker. Data on cfDNA integrity in differentiated thyroid cancer is sparse and needs to be investigated further. Aim of the study: The purpose of this study is to investigate the hypothesis that longer DNA strands in plasma may serve as a marker for thyroid cancer. Methods: Patients presenting with thyroid nodules underwent ultrasonography with Thyroid Image Reporting and Data Systems (TIRADS) scoring and FNAC (Bethesda classification).Patients with Bethesda 3, 4, 5, and 6 had surgery and confirmation by histopathology. Patients with Bethesda 2 presenting with compressive symptoms and cosmetic concerns underwent surgery; the remaining patients were considered benign based on clinical follow-up, USG, and FNAC characteristics. Blood sample collected prior to FNAC was used to extract and quantify cfDNA. To assess the integrity index 180/67, 306/67, and 476/67 for the APP gene, we used a quantitative real-time PCR (qPCR) method based on the quantification of four amplicons of varying lengths (67, 180, 306, and 476 bp, respectively). Because culture cells had extremely intact genomic DNA, the genomic DNA obtained from the MDA-T32 papillary thyroid cancer cell line was utilised as a reference to estimate the relative DNA strand integrity in plasma DNA. To determine the (ΔCt) value for 67,180, 306, and 476 bp, the Ct value of the sample was subtracted from that of the MDA-T32 control. Results: A total of 161 subjects were recruited, of these 92 were benign and 69 had differentiated thyroid cancer(confirmed by histopathology). Using the 180/67 bp integrity index benign thyroid nodule patients had significant lower value (median 0.39, 95% CI: 0.33-0.46) compared to patients with differentiated thyroid cancer (median 0.62, 95% CI: 0.56-0.68). A cfDNA integrity index of 180/67 bp could significantly differentiate between benign and malignant thyroid nodules (P<0.001). However, there was no significant difference between malignant and benign thyroid nodules based on the cell free DNA integrity indexes of 306/67 and 476/67. Conclusions: The cfDNA integrity score of 180/67 exhibits potential as a circulating marker for the detection of thyroid nodules and as a means of monitoring cfDNA fragmentation in patients with thyroid cancer. However, further confirmation through prospective trials is necessary. Presentation: 6/3/2024

8414 Utilization Of Cell Free DNA Integrity Index To Differentiate Benign From Malignant Thyroid Nodules

Abstract Disclosure: S. Tarafdar: None. S. Dutta: None. P. Mukhopadhyay: None. N.P. Bhattacharya: None. S. Ghosh: None. Introduction: Recent advances in molecular genetics have evaluated a potential role for plasma cell-free DNA (cfDNA) integrity index as a non-invasive cancer marker. Data on cfDNA integrity in differentiated thyroid cancer is sparse and needs to be investigated further. Aim of the study: The purpose of this study is to investigate the hypothesis that longer DNA strands in plasma may serve as a marker for thyroid cancer. Methods: Patients presenting with thyroid nodules underwent ultrasonography with Thyroid Image Reporting and Data Systems (TIRADS) scoring and FNAC (Bethesda classification).Patients with Bethesda 3, 4, 5, and 6 had surgery and confirmation by histopathology. Patients with Bethesda 2 presenting with compressive symptoms and cosmetic concerns underwent surgery; the remaining patients were considered benign based on clinical follow-up, USG, and FNAC characteristics. Blood sample collected prior to FNAC was used to extract and quantify cfDNA. To assess the integrity index 180/67, 306/67, and 476/67 for the APP gene, we used a quantitative real-time PCR (qPCR) method based on the quantification of four amplicons of varying lengths (67, 180, 306, and 476 bp, respectively). Because culture cells had extremely intact genomic DNA, the genomic DNA obtained from the MDA-T32 papillary thyroid cancer cell line was utilised as a reference to estimate the relative DNA strand integrity in plasma DNA. To determine the (ΔCt) value for 67,180, 306, and 476 bp, the Ct value of the sample was subtracted from that of the MDA-T32 control. Results: A total of 161 subjects were recruited, of these 92 were benign and 69 had differentiated thyroid cancer(confirmed by histopathology). Using the 180/67 bp integrity index benign thyroid nodule patients had significant lower value (median 0.39, 95% CI: 0.33-0.46) compared to patients with differentiated thyroid cancer (median 0.62, 95% CI: 0.56-0.68). A cfDNA integrity index of 180/67 bp could significantly differentiate between benign and malignant thyroid nodules (P<0.001). However, there was no significant difference between malignant and benign thyroid nodules based on the cell free DNA integrity indexes of 306/67 and 476/67. Conclusions: The cfDNA integrity score of 180/67 exhibits potential as a circulating marker for the detection of thyroid nodules and as a means of monitoring cfDNA fragmentation in patients with thyroid cancer. However, further confirmation through prospective trials is necessary. Presentation: 6/3/2024

High surface area cobalt aluminium layered double hydroxide printed electrodes for flexible supercapacitor and on-chip electrochemical bacterial lysing

This article describes the successful use of high surface area cobalt aluminium layered double hydroxide as an effective electrode material for fabricating highly flexible printed supercapacitor and electrochemical bacterial lysing chips. The flexible supercapacitor showed a high areal capacitance of 4.16 mF cm−2 at 0.4 mA cm−2 current density with 1.30 μWh cm−2 areal energy density and 301.04 μWcm−2 areal power density. Most importantly, the developed supercapacitor exhibited 91.32% initial areal capacitance even after 5000 continuous charge-discharge cycles and excellent mechanical stability over different bending states. An electrochemical chip for bacterial lysing was fabricated using a PDMS reservoir and catalyst-coated screen-printed carbon electrode. On-chip lysing of Gram-negative and Gram-positive bacteria was completely achieved at an applied potential of 3.5 V in 2 minutes, which is 1 V lower than that required for the unmodified electrode. The electrochemical chip outperforms commercial chemical lysing kits in terms of lysing time, DNA yield, longer DNA strands, and cost.

Multiomic Single-Cell Sequencing May Distinguish BCR-ABL-Mutated Acute Myeloid Leukemia (AML) from Blast Crisis Chronic Myelogenous Leukemia (BC-CML)

Introduction The existence of BCR-ABL+ AML as an entity distinct from BC-CML is controversial. The WHO recently recognized BCR-ABL+ AML, but with the caveat that it is poorly characterized (Khoury, 2022). Clinically, BCR-ABL+ AML is associated with high risk of relapse (Neuendorff, 2018). Further, acquired BCR-ABL mutations have been identified as a cause of relapse on FLT3 inhibitors (McMahon, 2019). We hypothesize that BCR-ABL+ AML can be distinguished as a distinct biologic entity from BC-CML by the observation of BCR-ABL as a sub-clonal rather than a founder mutation in leukemogenesis. Methods To test the utility of multi-omic single cell (SC) sequencing in determining tumor phylogeny and identification of BCR-ABL+ AML vs BC-CML, we analyzed bone marrow samples from 4 patients (pts): 1 relapsed AML with clinical development of a new BCR-ABL fusion at the time of relapse, and 3 newly diagnosed BC-CML (2 myeloid, 1 lymphoid). We performed high-throughput SC DNA sequencing with simultaneous cell-surface immunophenotyping and targeted RNA sequencing for BCR-ABL fusion proteins (Mission Bio Tapestri). All SC DNA sequencing included pt-specific primers for BCR-ABL DNA breakpoints, which were previously identified using nanopore-based technology for sequencing long strands of DNA. Results We analyzed 19,197 single cells (median 1995 cells/pt). Multi-omic SC technology differentiated BCR-ABL+ AML from BC-CML by confirming the fusion arose as a branch mutation from a founding leukemic clone and was not present in all leukemic populations. In Patient 1, who was sampled at time of AML relapse, we identified pathogenic FLT3 ITD and NRAS Q61K mutations. FLT3 co-mutated with either the BCR-ABL DNA breakpoint or NRAS in the same single cell and clone, while the BCR-ABL DNA breakpoint and NRAS were not co-mutated. Phylogenetic reconstruction determined FLT3 to be a founding mutation, which later branched into FLT3/BCR-ABL and FLT3/ NRAS co-mutant subclones. Further, the FLT3-ITD/BCR-ABL and FLT3-ITD/ NRAS subclones had distinct immunophenotypes. Blasts harboring the BCR-ABL DNA breakpoint were predominantly identified in the CD117+/CD33+ population (60.2% of cells) compared to CD34+ (16.4% of cells, p <0.001) and blasts with monocytic differentiation (19.1% of cells, p = 0.02). Notably, BCR-ABL+ cells were not identified in T cells. NRAS mutations were predominantly identified in both CD34+ and monocytic blasts. Together, these data support BCR-ABL arising late within the evolutionary history of this pt's AML. By contrast, in Patients 2 and 3 with myeloid BC-CML, the BCR-ABL DNA breakpoint and corresponding BCR-ABL fusion were detectable in all cell populations, including blasts as well as immunophenotypically normal T cells, B cells, and macrophages. In Patient 2, we identified an IDH2 R132C mutation co-mutated with BCR-ABL. Phylogenetic reconstruction determined the IDH2 mutation arose as a branch from a founding BCR-ABL+clone. Unlike the BCR-ABL breakpoint, which was present in all cell populations, the IDH2 mutation was only identified in immunophenotypically-defined CD33+/CD34+/CD117+ myeloid blasts and was absent from bystander T cells and macrophages. Finally, Patient 4had a clinical diagnosis of lymphoid BC-CML. Like Patients 2 and 3, the BCR-ABL DNA breakpoint and corresponding RNA fusion were present in all immunophenotypically-defined blast populations. Unlike Patients 2 and 3, however, BCR-ABL was absent from immunophenotypically normal CD4+ and CD8+ T cells, macrophages, and monocytes. Additional pts are needed to determine whether this pattern is consistent with all lymphoid BC-CML or whether this pt's biology may have been closer to BCR-ABL+ acute lymphoblastic leukemia (ALL). Conclusions Multi-omic SC technology can distinguish BCR-ABL+ AML from myeloid BC-CML and provide direct confirmation that lineage-restriction of BCR-ABL potentially clarifies the diagnosis of discrete Ph+ leukemias. Future studies are needed to understand the role of RT-PCR based BCR-ABL1 testing as a marker of minimal residual disease in pts with BCR-ABL+ subclones, the impact of BCR-ABL as a potential driver of AML resistance, and the utility of BCR-ABL inhibitors in leukemias with late-arising BCR-ABL+ clones. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org. Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224

Accurate gene consensus at low nanopore coverage.

Nanopore technologies allow high-throughput sequencing of long strands of DNA at the cost of a relatively large error rate. This limits its use in the reading of amplicon libraries in which there are only a few mutations per variant and therefore they are easily confused with the sequencing noise. Consensus calling strategies reduce the error but sacrifice part of the throughput on reading typically 30 to 100 times each member of the library. In this work, we introduce SINGLe (SNPs In Nanopore reads of Gene Libraries), an error correction method to reduce the noise in nanopore reads of amplicons containing point variations. SINGLe exploits that in an amplicon library, all reads are very similar to a wild-type sequence from which it is possible to experimentally characterize the position-specific systematic sequencing error pattern. Then, it uses this information to reweight the confidence given to nucleotides that do not match the wild-type in individual variant reads and incorporates it on the consensus calculation. We tested SINGLe in a mutagenic library of the KlenTaq polymerase gene, where the true mutation rate was below the sequencing noise. We observed that contrary to other methods, SINGLe compensates for the systematic errors made by the basecallers. Consequently, SINGLe converges to the true sequence using as little as 5 reads per variant, fewer than the other available methods.

Physical Nature of Chromatin in the Nucleus.

Genomic information is encoded on long strands of DNA, which are folded into chromatin and stored in a tiny nucleus. Nuclear chromatin is a negatively charged polymer composed of DNA, histones, and various nonhistone proteins. Because of its highly charged nature, chromatin structure varies greatly depending on the surrounding environment (e.g., cations, molecular crowding, etc.). New technologies to capture chromatin in living cells have been developed over the past 10 years. Our view on chromatin organization has drastically shifted from a regular and static one to a more variable and dynamic one. Chromatin forms numerous compact dynamic domains that act as functional units of the genome in higher eukaryotic cells and locally appear liquid-like. By changing DNA accessibility, these domains can govern various functions. Based on new evidences from versatile genomics and advanced imaging studies, we discuss the physical nature of chromatin in the crowded nuclear environment and how it is regulated.

Self-Assembly of DNA-Functionalized Nanoparticles Guided by Binding Kinetics.

We use a coarse-grained model of DNA-functionalized particles (DFPs) to understand the role of DNA strand length on their self-assembly. We find that the increasing strand length for a given particle size decreases the propensity to form ordered crystalline assemblies within the simulation time. Instead, disordered structures form when the strand length exceeds a certain threshold, consistent with the previous experiments. Analysis of the simulation data based on a pair of DFPs suggests weakening interparticle interactions with increasing strand length, thereby shifting the suitable assembly conditions to lower temperatures. We find that DNA (un)hybridization kinetics at these lower temperatures becomes significantly slower, preventing systems with longer DNA strands from crystallizing successfully. We suggest that a suitable strategy to overcome this kinetic barrier is to enhance interparticle interactions for DFPs with longer DNA strands, which is achieved by increasing the DNA grafting density. We directly test this hypothesis and show successful crystallization within the simulation time for DFPs with longer strands with higher grafting densities. Our results highlight the power of computational modeling in elucidating the fundamental design principles and guiding the assembly of nanoparticles to form complex nanostructures in cases where experiments alone have not been able to do so.

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and non-cancerous cells.

A scalable, efficient, and safe method to prepare high quality DNA from mycobacteria and other challenging cells

The rapid development in sequencing technology is creating an increase in demand for largely intact DNA as starting material as very long strands of DNA are sequenced directly to generate reads that are thousands of bases long. Organisms with thick cell walls are difficult to lyse, often impacting both DNA recovery and quality. Consequently, most mycobacterial DNA extraction methods require bead-beating steps or toxic chemicals. Here we present an updated method that yields abundant, high quality genomic DNA from M. tuberculosis and diverse nontuberculous mycobacterial (NTM) species, in addition to complex biological communities from a variety of sources. This method eliminates the time-consuming phenol and chloroform extraction and ethanol precipitation steps, and high quality DNA from up to 96 samples can be extracted in about 2–3 h of hands-on time. This DNA is suitable for long and short read sequencing technologies as well as PCR and qPCR amplification.

COMPARATIVE CHARACTERISTIC OF EFFECTIVENESS OF SOME DECONTAMINATION FACILITIES USED FOR CARRYING OUT OF FORENSIC MOLECULAR GENETIC EXAMINATION

The article is devoted to comparing the performance of disinfectants at different concentrations of a contaminating agent. Contamination of objects can occur at any stage of the study, so effective methods of decontamination should be used to prevent it, which at the same time should be safe for laboratory staff. The purpose of the work is to check the effectiveness of disinfectants while working in biological laboratories and to determine their effects on the genetic information of the cell, the effect on the inhibition of PCR reaction and DNA degradation. The mechanisms of influence on a cell and DNA of chemical solutions are analyzed: Винсепт експресс, Бациллол АФ, Дезактин 0, 2 %, WIP'ANIOS Premium, DNAZap™ PCR DNA Degradation Solutions, 70 % и 96 % ethyl alcohols, and physical methods: ultraviolet radiation, alcohol treatment followed by calcination in the flame of the burner and treatment with bidistilled deionized water. While research the contaminating DNA is represented by the synthetic genomic DNA produced by the Promega company. As a result of the pilot study, DNAZap ™ PCR DNA Degradation Solutions and bidistilled deionized water had the highest decontamination efficiency. Bidistilled deionized water can be used to the fullest in the laboratory if medical waste disposal standards are met. Other methods showed an efficiency of more than 99.98% which also allows them to be used when working in a biological laboratory, following the suggested recommendations. The presented methods of decontamination did not cause inhibition of the PCR reaction. Preserved, after decontamination treatment, DNA for most agents had a low level of degradation. After being treated with ultraviolet radiation, the level of DNA degradation was medium which means that ultraviolet radiation destroys long strands of DNA, leaving strands unbroken up to 200 bp. In addition, increasing the concentration of contaminating DNA did not affect the change in the efficiency of decontamination substances. All the decontaminated substances analyzed can be modified in molecular genetic laboratories.

Trapping DNA with a high throughput microfluidic device

Long strands of DNA can be trapped and concentrated near the inlet of a microfluidic channel by applying a pressure gradient and an opposing electric field. The mechanism for trapping involves a migration of DNA perpendicular to both the fluid flow and the electric field. Migration leads to a highly nonuniform distribution of DNA within a cross section of the channel, with the bulk of the DNA concentrated in a thin (10 μm) layer next to the walls of the channel. This highly concentrated layer generates an electrophoretic flux toward the inlet to the device, despite the much larger fluid flow in the opposite direction. In this paper, the extent to which DNA can be trapped and concentrated by this means has been characterized by fluorescence measurements. At short times (<2 hours) nearly all the incoming DNA remains trapped within the device until the electric field is turned off. The DNA largely accumulates near the inlet, but after 30-60 minutes additional DNA starts to accumulate deeper into the channel. Eventually DNA leaks from the device itself, but ≈80% of the incoming DNA can be retained for up to 5 hours. Optimizing the electric field strength can increase the amount of DNA that can be trapped, but the efficiency is not affected by the channel cross-section.

Microfluidics platform simplifies preparation of long DNA strands for optical mapping

New technique using alginate gel microparticles promises to provide easier use of benchtop techniques for handling long strands of DNA from single-cell samples.

Deformation Modes of Giant Unilamellar Vesicles Encapsulating Biopolymers.

The shapes of giant unilamellar vesicles (GUVs) enclosing polymer molecules at relatively high concentration, used as a model cytoplasm, significantly differ from those containing only small molecules. Here, we investigated the effects of the molecular weights and concentrations of polymers such as polyethylene glycol (PEG), bovine serum albumin (BSA), and DNA on the morphology of GUVs deflated by osmotic pressure. Although small PEG (MW < 1000) does not alter the mode of shape transformation even at >10% (w/w), PEG with MW > 6000 induces budding and pearling transformation at above 1% (w/w). Larger PEG frequently induced small buddings and tubulation from the membrane of mother GUVs. A similar trend was observed with BSA, indicating that the effect is irrelevant to the chemical nature of polymers. More surprisingly, long strands of DNA (>105 bp) enclosed in GUVs induced budding transformation at concentrations as low as 0.01-0.1% (w/w). We expect that this molecular size dependency arises mainly from the depletion volume effect. Our results showed that curving, budding, and tubulation of lipid membranes, which are ubiquitous in living cells, can result from simple cell-mimics consisting of the membrane and cytosolic macromolecules, but without specific shape-determining proteins.

Rotation-Induced Macromolecular Spooling of DNA

Genetic information is stored in a linear sequence of base-pairs; however, thermal fluctuations and complex DNA conformations such as folds and loops make it challenging to order genomic material for in vitro analysis. In this work, we discover that rotation-induced macromolecular spooling of DNA around a rotating microwire can monotonically order genomic bases, overcoming this challenge. We use single-molecule fluorescence microscopy to directly visualize long DNA strands deforming and elongating in shear flow near a rotating microwire, in agreement with numerical simulations. While untethered DNA is observed to elongate substantially, in agreement with our theory and numerical simulations, strong extension of DNA becomes possible by introducing tethering. For the case of tethered polymers, we show that increasing the rotation rate can deterministically spool a substantial portion of the chain into a fully stretched, single-file conformation. When applied to DNA, the fraction of genetic information sequentially ordered on the microwire surface will increase with the contour length, despite the increased entropy. This ability to handle long strands of DNA is in contrast to modern DNA sample preparation technologies for sequencing and mapping, which are typically restricted to comparatively short strands resulting in challenges in reconstructing the genome. Thus, in addition to discovering new rotation-induced macromolecular dynamics, this work inspires new approaches to handling genomic-length DNA strands.

"Biodrop" Evaporation and Ring-Stain Deposits: The Significance of DNA Length.

Small sessile drops of water containing either long or short strands of DNA ("biodrops") were deposited on silicon substrates and allowed to evaporate. Initially, the triple line (TL) of both types of droplet remained pinned but later receded. The TL recession mode continued at constant speed until almost the end of drop lifetime for the biodrops with short DNA strands, whereas those containing long DNA strands entered a regime of significantly lower TL recession. We propose a tentative explanation of our observations based on free energy barriers to unpinning and increases in the viscosity of the base liquid due to the presence of DNA molecules. In addition, the structure of DNA deposits after evaporation was investigated by AFM. DNA self-assembly in a series of perpendicular and parallel orientations was observed near the contact line for the long-strand DNA, while, with the short-stranded DNA, smoother ring-stains with some nanostructuring but no striations were evident. At the interior of the deposits, dendritic and faceted crystals were formed from short and long strands, respectively, due to diffusion and nucleation limited processes, respectively. We suggest that the above results related to the biodrop drying and nanostructuring are indicative of the importance of DNA length, i.e., longer DNA chains consisting of linearly bonded shorter, rod-like DNA strands.

Steps for Constructing Synthetic Membrane Curvature-Inducing DNA Origami Scaffolds

Biological membranes are dynamic cellular barriers that suffer deformation and bending. Despite huge effort in identifying the physical-chemical fundaments of membrane curvature, the particular mechanisms and elements of curvature induction on which cells actually base their membrane transformations are still poorly understood. In this project, we aim to fill this gap by engineering a minimal membrane curvature-inducing scaffold.Due to its exclusive nano-engineering properties, the DNA origami technology was chosen to build synthetic curvature-inducing scaffolds. This state-of-the-art technology enables the folding of long strands of DNA into nano-objects with defined shapes by using sequence-specific short DNA staples. Here, we designed DNA nanostructures with defined curvature and shape, as well as specific membrane binding positions. Hybrid origami scaffolds with specific functional membrane-attachment groups have been produced and the interaction of those hybrid scaffolds with lipid membrane model systems was studied, more precisely in terms of localization, membrane density and induction of membrane curvature.By using fluorescence microscopy, we show that the positioning and number of the membrane binding elements are essential to ensure an effective localization of the DNA nanostructures to the membrane. Moreover, the requirements seem to be dependent on the intrinsic curvature of the nanostructures. Interestingly, the quantitative characterization of our minimal membrane-scaffolds through fluorescence correlation spectroscopy (FCS) unravels that they may bind cooperatively to the membrane. In the end, our approach aims to reveal the minimal set of modules needed for shape-dependent induction of membrane curvature.In this work, we have developed and characterized the membrane binding properties of a synthetic membrane scaffold, which can act as biomimetic of the properties of biological scaffolding elements.

High-throughput optical sensing of nucleic acids in a nanopore array.

Protein nanopores such as α-hemolysin and MspA can potentially be used to sequence long strands of DNA quickly and at low cost. In order to provide high-speed sequencing, large arrays of nanopores are required that allow the nanopores to individually addressed, but current nanopore sequencing methods rely on ionic current measurements and such methods are likely to prove difficult to scale up. Here, we show that, by optically encoding the ionic flux through protein nanopores, the discrimination of nucleic acid sequences and the detection of sequence-specific nucleic acid binding events can be parallelized. We make optical recordings at a density of ~104 nanopores per mm2 in a single droplet interface bilayer. Nanopore blockades can discriminate between DNAs with sub-pA equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. By creating an array of 2500 bilayers with a micro-patterned hydrogel chip, we are also able to load different samples into specific bilayers, suitable for high-throughput nanopore recording.

Feasibility of Population Scale Comprehensive Identification and Analysis of Complex Structural Variations in Cancer Genome Using Nanochannel Array

Diseases with complex traits such as hematological cancer are known to be associated with large structural variations (SV > 1 kb). Techniques such as karyotyping, FISH and aCGH have been common tools for gross chromosomal lesion analysis. Yet population scale comprehensive SV analysis with these tools remains impractical, tedious or incomplete such as missing balanced lesions by aCGH. Recently, high throughput Next Generation Sequencing (NGS) has generated a large amount of sequencing reads that have rapidly reduced costs and are effective in detection of SNPs and small indels; however, complicated by the fact that large structural variations, often spanning tens to hundreds of thousands of base pairs or involving complex rearrangements throughout the genome, are hard for current short read sequencing technology to assemble or infer. Therefore, there is a blind spot in effectively detecting SVs within this range (1 kb ~ 1 Mb) due to insufficient tools.Here we demonstrate a technology that rapidly linearizes very long strands of genomic DNA (100 kb to Mbs) through NanoChannels to directly visualize large SVs and rearrangements preserved within intact genomic DNA at the single molecule level. Through specific sequence motif labeling, de novo genome physical maps are assembled within hours and hundreds to thousands of SV events, balanced or imbalanced, are called with no a priori knowledge of the samples. Preliminary testing data from a group of cancer samples and multiple trio families will be shown to demonstrate this highly comprehensive and cost effective approach, with results validated by direct single-molecule images and multiple orthogonal methods. We also show long spanning molecules would provide very valuable information of precise mapping of viral genomic sequence integration in human genomes that are potentially associated with malignant cell transformation. For the first time, it is now feasible to do large population-based comprehensive genome structural variation studies on a single platform. This innovations will transform the biomedical research, diagnosis and treatment of hematological cancers that result from structural variations and chromosomal lesions. DisclosuresNo relevant conflicts of interest to declare.

Synthetic Membrane Curvature-Inducing DNA Origami Scaffolds

Biological membranes are dynamic cellular barriers that suffer deformation and bending. Despite huge effort in identifying the general elements involved in membrane curvature, the physical-chemical basis of curvature induction is still poorly understood. In this work, we fill this gap by engineering a minimal curvature-inducing system. Due to its exclusive nanoengineering properties, DNA origami technology will be utilized to build minimal curvature-inducing scaffoldings. This state-of-the-art technology enables the folding of long strands of DNA into nano-objects with defined shapes by using sequence-specific short DNA staples. For instance, our group recently constructed membrane-interacting rod-like DNA origami structures, which were functionalized with hydrophobic anchors and fluorescently labeled at defined positions [1]. Here, synthetic nanostructures will be designed in order to have a) defined customized shapes and b) specific membrane binding elements. Hybrid origami scaffolds with specific functional membrane-attachment groups bound at defined positions on the scaffolds will be produced. The interaction of those hybrid scaffolds with lipid membrane model systems will be studied and their capability of inducing membrane curvature evaluated. Fluorescence microscopy and atomic force microscopy methods will be used in order to retrieve extent, localization and forces involved in the interactions. More precisely, the role of the scaffolding shapes, membrane-attachment moieties, oligomerization and conformational changes will be assessed. At the end, this quantitative characterization of minimal membrane-inducing scaffolds will help understand the role of cooperativity in membrane deformation and the rules that govern the induction of membrane curvature.[1] A. Czogalla, E. P. Petrov, D. J. Kauert, V. Uzunova, Y. Zhang, R. Seidel, and P. Schwille, Faraday Discuss. 161 (2013) 31.

DNA sequencing using electrical conductance measurements of a DNA polymerase

The development of personalized medicine-in which medical treatment is customized to an individual on the basis of genetic information-requires techniques that can sequence DNA quickly and cheaply. Single-molecule sequencing technologies, such as nanopores, can potentially be used to sequence long strands of DNA without labels or amplification, but a viable technique has yet to be established. Here, we show that single DNA molecules can be sequenced by monitoring the electrical conductance of a phi29 DNA polymerase as it incorporates unlabelled nucleotides into a template strand of DNA. The conductance of the polymerase is measured by attaching it to a protein transistor that consists of an antibody molecule (immunoglobulin G) bound to two gold nanoparticles, which are in turn connected to source and drain electrodes. The electrical conductance of the DNA polymerase exhibits well-separated plateaux that are ~3 pA in height. Each plateau corresponds to an individual base and is formed at a rate of ~22 nucleotides per second. Additional spikes appear on top of the plateaux and can be used to discriminate between the four different nucleotides. We also show that the sequencing platform works with a variety of DNA polymerases and can sequence difficult templates such as homopolymers.