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Rxivist combines biology preprints from bioRxiv and medRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 128,741 papers from 551,614 authors.

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in category molecular biology

3,856 results found. For more information, click each entry to expand.

61: RNA polymerase II clustering through CTD phase separation
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Posted 07 May 2018

RNA polymerase II clustering through CTD phase separation
3,709 downloads bioRxiv molecular biology

M. Boehning, C. Dugast-Darzacq, M. Rankovic, A. S. Hansen, T. Yu, H. Marie-Nelly, D.T. McSwiggen, G. Kokic, G. M. Dailey, P. Cramer, X. Darzacq, M. Zweckstetter

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

62: Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
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Posted 14 Nov 2018

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
3,681 downloads bioRxiv molecular biology

Beeke Wienert, Stacia K. Wyman, Christopher D Richardson, Charles D Yeh, Pinar Akcakaya, Michelle J. Porritt, Michaela Morlock, Jonathan T. Vu, Katelynn R. Kazane, Hannah L Watry, Luke M Judge, Bruce R. Conklin, Marcello Maresca, Jacob E. Corn

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.

63: Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro
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Posted 14 Apr 2020

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro
3,675 downloads bioRxiv molecular biology

Haixia Su, Sheng Yao, Wenfeng Zhao, Minjun Li, Jia Liu, WeiJuan Shang, Hang Xie, Changqiang Ke, Meina Gao, Kunqian Yu, Hong Liu, Jingshan Shen, Wei Tang, Leike Zhang, Jianping Zuo, Ruotian Jiang, Fang Bai, Yan Wu, Yang Ye, Yechun Xu

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a 'shield' in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs. ### Competing Interest Statement The authors have declared no competing interest.

64: The SARS-CoV-2 S1 spike protein mutation N501Y alters the protein interactions with both hACE2 and human derived antibody: A Free energy of perturbation study
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Posted 26 Dec 2020

The SARS-CoV-2 S1 spike protein mutation N501Y alters the protein interactions with both hACE2 and human derived antibody: A Free energy of perturbation study
3,661 downloads bioRxiv molecular biology

Filip Fratev

The N501Y and K417N mutations in spike protein of SARS-CoV-2 and their combination arise questions but the data about their mechanism of action at molecular level is limited. Here, we present Free energy perturbation (FEP) calculations for the interactions of the spike S1 receptor binding domain (RBD) with both the ACE2 receptor and an antibody derived from COVID-19 patients. Our results shown that the S1 RBD-ACE2 interactions were significantly increased whereas those with the STE90-C11 antibody dramatically decreased; about over 100 times. The K417N mutation had much more pronounced effect and in a combination with N501Y fully abolished the antibody effect. This may explain the observed in UK and South Africa more spread of the virus but also raise an important question about the possible human immune response and the success of already available vaccines.

65: Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody
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Posted 10 Jun 2020

Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity Nanobody
3,623 downloads bioRxiv molecular biology

Guillermo Valenzuela Nieto, Ronald Jara, Daniel Watterson, Naphak Modhiran, Alberto A Amarilla, Johanna Himelreichs, Alexander A Khromykh, Constanza Salinas, Teresa Pinto, Yorka Cheuquemilla, Yago Margolles, Natalia López González del Rey, Zaray Miranda-Chacon, Alexei Cuevas, Anne Berking, Camila Deride, Sebastián González-Moraga, Héctor Mancilla, Daniel Maturana, Andreas Langer, Juan Pablo Toledo, Ananda Müller, Benjamín Uberti, Paola Krall, Pamela Ehrenfeld, Javier Blesa, Pedro Chana-Cuevas, German Rehren, David Schwefel, Luis Ángel Fernandez, Alejandro Rojas-Fernandez

Despite unprecedented global efforts to rapidly develop SARS-CoV-2 treatments, in order to reduce the burden placed on health systems, the situation remains critical. Effective diagnosis, treatment, and prophylactic measures are urgently required to meet global demand: recombinant antibodies fulfill these requirements and have marked clinical potential. Here, we describe the fast-tracked development of an alpaca Nanobody specific for the receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein with therapeutic potential applicability. We present a rapid method for nanobody isolation that includes an optimized immunization regimen coupled with VHH library E. coli surface display, which allows single-step selection of high-affinity nanobodies using a simple density gradient centrifugation of the bacterial library. The selected single and monomeric Nanobody, W25, binds to the SARS-CoV-2 S RBD with sub-nanomolar affinity and efficiently competes with ACE-2 receptor binding. Furthermore, W25 potently neutralizes SARS-CoV-2 wild type and the D614G variant with IC50 values in the nanomolar range, demonstrating its potential as antiviral agent. ### Competing Interest Statement Conflict of interest statement The Austral University of Chile claiming priority to U.S. Provisional Patent Application No. US Serial No. 63/025534, filed MAY-2020.

66: Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing
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Posted 16 Apr 2019

Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing
3,607 downloads bioRxiv molecular biology

Heather L Drexler, Karine Choquet, L. Stirling Churchman

Human genes have numerous exons that are differentially spliced within pre-mRNA. Understanding how multiple splicing events are coordinated across nascent transcripts requires quantitative analyses of transient RNA processing events in living cells. We developed nanopore analysis of CO-transcriptional Processing (nano-COP), in which nascent RNAs are directly sequenced through nanopores, exposing the dynamics and patterns of RNA splicing without biases introduced by amplification. nano-COP showed that in both human and Drosophila cells, co-transcriptional splicing occurs after RNA polymerase II transcribes several kilobases of pre-mRNA, suggesting that metazoan splicing transpires distally from the transcription machinery. Inhibition of the branch-site recognition complex SF3B globally abolished co-transcriptional splicing in both species. Our findings revealed that splicing order does not strictly follow the order of transcription and is influenced by cis-regulatory elements. In human cells, introns with delayed splicing frequently neighbor alternative exons and are associated with RNA-binding factors. Moreover, neighboring introns in human cells tend to be spliced concurrently, implying that splicing occurs cooperatively. Thus, nano-COP unveils the organizational complexity of metazoan RNA processing.

67: Sub-tomogram averaging in RELION
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Posted 04 Nov 2015

Sub-tomogram averaging in RELION
3,534 downloads bioRxiv molecular biology

Tanmay A.M. Bharat, Sjors Scheres

Electron cryo-tomography (cryo-ET) and sub-tomogram averaging allow structure determination of macromolecules in situ, and are gaining in popularity for initial model generation for single- particle analysis. We describe herein, a protocol for sub-tomogram averaging from cryo-ET data using the RELION software. We describe how to calculate newly developed three-dimensional models for the contrast transfer function and the missing wedge of each sub-tomogram, and how to use these models for regularized-likelihood refinement. This approach has been implemented in the existing workflow for single-particle analysis, so that users may conveniently tap into existing capabilities of the RELION software. As example applications, we present analyses of purified hepatitis B capsid particles and S. cerevisiae 80S ribosomes. In both cases, we show that following initial classification, sub-tomogram averaging in RELION allows de novo generation of initial models, and provides high-resolution maps where secondary structure elements are resolved.

68: ChIP-seq of plasma cell-free nucleosomes identifies cell-of-origin gene expression programs
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Posted 15 May 2019

ChIP-seq of plasma cell-free nucleosomes identifies cell-of-origin gene expression programs
3,431 downloads bioRxiv molecular biology

Ronen Sadeh, Israa Sharkia, Gavriel Fialkoff, Ayelet Rahat, Jenia Gutin, Alon Chappleboim, Mor Nitzan, Ilana Fox-Fisher, Daniel Neiman, Guy Meler, Zahala Kamari, Dayana Yaish, Tamar Peretz, Ayala Hubert, Jonatan E Cohen, Salach Azzam, Mark Temper, Albert Grinshpun, Myriam Maoz, Samir Abu-Gazala, Ami Ben Ya’acov, Eyal Shteyer, Rifaat Safadi, Tommy Kaplan, Ruth Shemer, David Planer, Eithan Galun, Benjamin Glaser, Aviad Zick, Yuval Dor, Nir Friedman

Genomic DNA is packed by histone proteins that carry a multitude of post-translational modifications that reflect cellular transcriptional state. Cell-free DNA (cfDNA) is derived from fragmented chromatin in dying cells, and as such it retains the histones markings present in the cells of origin. Here, we pioneer chromatin immunoprecipitation followed by sequencing of cell-free nucleosomes (cfChIP-seq) carrying active chromatin marks. Our results show that cfChIP-seq provides multidimensional epigenetic information that recapitulates the epigenetic and transcriptional landscape in the cells of origin. We applied cfChIP-seq to 268 samples including samples from patients with heart and liver pathologies, and 135 samples from 56 metastatic CRC patients. We show that cfChIP-seq can detect pathology-related transcriptional changes at the site of the disease, beyond the information on tissue of origin. In CRC patients we detect clinically-relevant, and patient-specific information, including transcriptionally active HER2 amplifications. cfChIP-seq provides genome-wide information and requires low sequencing depth. Altogether, we establish cell-free chromatin immunoprecipitation as an exciting modality with potential for diagnosis and interrogation of physiological and pathological processes using a simple blood test. One Sentence Summary ChIP-seq of plasma-circulating nucleosomes (cfChIP-seq) from a simple blood test provides detailed information about gene expression programs in human organs, and cancer. ### Competing Interest Statement A patent application for cfChIP-seq has been submitted by the Hebrew University of Jerusalem.

69: Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin
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Posted 21 Jun 2019

Super-resolution Imaging Reveals 3D Structure and Organizing Mechanism of Accessible Chromatin
3,408 downloads bioRxiv molecular biology

Liangqi Xie, Peng Dong, Yifeng Qi, Margherita De Marzio, Xingqi Chen, Sambashiva Banala, Wesley R Legant, Brian P. English, Anders S. Hansen, Anton Schulmann, Luke D. Lavis, Eric Betzig, Rafael Casellas, Howard Y. Chang, Bin Zhang, Robert Tjian, Zhe Liu

Access to cis-regulatory elements packaged in chromatin is essential for directing gene expression and cell viability. Here, we report a super-resolution imaging strategy, 3D ATAC-PALM, that enables direct visualization of the entire accessible genome. We found that active chromosomal segments are organized into spatially-segregated accessible chromatin domains (ACDs). Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced enhanced ACD clustering, reduced physical separation between intrachromosomal ACDs, and differentially regulated ACD compaction. Experimental perturbations and polymer modeling suggest that dynamic protein-protein and protein-DNA interactions within ACDs couple with loop extrusion to organize ACD topology. Dysorganization of ACDs upon CTCF or Cohesin loss alters transcription factor binding and target search dynamics in living cells. These results uncover fundamental mechanisms underpinning the formation of 3D genome architecture and its pivotal function in transcriptional regulation.

70: Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing
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Posted 06 Nov 2014

Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing
3,388 downloads bioRxiv molecular biology

Catherine M Burke, Aaron E. Darling

We describe a method for sequencing full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform. The resulting sequences have about 100-fold higher accuracy than standard Illumina reads and are chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. We demonstrate that the data provides fine scale phylogenetic resolution not available from Illumina amplicon methods targeting smaller variable regions of the 16S rRNA gene.

71: Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing
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Posted 23 Sep 2017

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing
3,296 downloads bioRxiv molecular biology

Matthew B Begemann, Benjamin N Gray, Emma January, Anna Singer, Dylan C Kesler, Yonghua He, Haijun Liu, Hongjie Guo, Alex Jordan, Thomas P. Brutnell, Todd C Mockler, Mohammed Oufattole

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

72: Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning
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Posted 30 Apr 2019

Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning
3,280 downloads bioRxiv molecular biology

Eric M Jones, Nathan B. Lubock, AJ Venkatakrishnan, Jeffrey Wang, Alex M Tseng, Joseph Paggi, Naomi R Latorraca, Daniel Cancilla, Megan Satyadi, Jessica E Davis, M. Madan Babu, Ron O. Dror, Sriram Kosuri

In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

73: P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells
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Posted 26 Jul 2017

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells
3,275 downloads bioRxiv molecular biology

Robert J Ihry, Kathleen A Worringer, Max R Salick, Elizabeth Frias, Daniel Ho, Kraig Theriault, Sravya Kommineni, Julie Chen, Marie Sondey, Chaoyang Ye, Ranjit Randhawa, Tripti Kulkarni, Zinger Yang, Gregory McAllister, Carsten Russ, John Reece-Hoyes, William Forrester, Gregory R Hoffman, Ricardo Dolmetsch, Ajamete Kaykas

CRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response is tp53-dependent and the toxic effect of tp53 severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor for tp53 function, especially in hPSCs which spontaneously acquire tp53 mutations.

74: High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations
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Posted 01 Mar 2018

High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations
3,271 downloads bioRxiv molecular biology

Jason M. Gehrke, Oliver Cervantes, M. Kendell Clement, Luca Pinello, J. Keith Joung

Recently described base editor (BE) technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine (C to T) DNA alterations in many different cell types and organisms. In contrast to genome-editing nucleases, BEs avoid the need to introduce double-strand breaks or exogenous donor DNA templates and induce lower levels of unwanted variable-length insertion/deletion mutations (indels). However, existing BEs can also efficiently create unwanted C to T alterations when more than one C is present within the five base pair "editing window" of these proteins, a lack of precision that can cause potentially deleterious bystander mutations. Mutations in the cytidine deaminase enzyme can shorten the length of the editing window and thereby partially address this limitation but these BE variants still do not discriminate among multiple cytidines within the narrowed window and also possess a more limited targeting range. Here, we describe an alternative strategy for reducing bystander mutations using a novel BE architecture that harbors an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines according to a TCR>TCY>VCN (V = G, A, C, Y = C, T) hierarchy. In direct comparisons with the widely used BE3 fusion in human cells, our eA3A-BE3 fusion exhibits comparable activities on cytidines in TC motifs but greatly reduced or no significant editing on cytidines in other sequence contexts. Importantly, we show that eA3A-BE3 can correct a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3, substantially minimizing the creation of an undesirable bystander mutation. Surprisingly, we also found that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites. Our results validate a general strategy to improve the precision of base editors by engineering their cytidine deaminases to possess greater sequence specificity, an important proof-of-principle that should motivate the development of a larger suite of new base editors with such properties.

75: CryoDRGN: Reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks
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Posted 29 Mar 2020

CryoDRGN: Reconstruction of heterogeneous structures from cryo-electron micrographs using neural networks
3,242 downloads bioRxiv molecular biology

Ellen D. Zhong, Tristan Bepler, Bonnie Berger, Joseph H. Davis

Cryo-EM single-particle analysis has proven powerful in determining the structures of rigid macromolecules. However, many protein complexes are flexible and can change conformation and composition as a result of functionally-associated dynamics. Such dynamics are poorly captured by current analysis methods. Here, we present cryoDRGN, an algorithm that for the first time leverages the representation power of deep neural networks to efficiently reconstruct highly heterogeneous complexes and continuous trajectories of protein motion. We apply this tool to two synthetic and three publicly available cryo-EM datasets, and we show that cryoDRGN provides an interpretable representation of structural heterogeneity that can be used to identify discrete states as well as continuous conformational changes. This ability enables cryoDRGN to discover previously overlooked structural states and to visualize molecules in motion.

76: Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites
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Posted 22 Jan 2016

Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites
3,237 downloads bioRxiv molecular biology

David L. Stern

Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence reads that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor sequence generates oriented reads that flank and are oriented toward the transposable element insertion site. The convergent orientation of adjacent reads at the insertion site allows straightforward prediction of the precise insertion site(s). A Linux shell script is provided to identify insertion sites from fastq files.

77: A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA
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Posted 27 Jan 2018

A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA
3,181 downloads bioRxiv molecular biology

Tomas Klingström, Erik Bongcam-Rudloff, Olga Vinnere Pettersson

During DNA extraction the DNA molecule undergoes physical and chemical shearing, causing the DNA to fragment into shorter and shorter pieces. Under common laboratory conditions this fragmentation yields DNA fragments of 5-35 kilobases (kb) in length. This fragment length is more than sufficient for DNA sequencing using short-read technologies which generate reads 50-600 bp in length, but insufficient for long-read sequencing and linked reads where fragment lengths of more than 40 kb may be desirable. This study provides a theoretical framework for quality management to ensure access to high molecular weight DNA in samples. Shearing can be divided into physical and chemical shearing which generate different patterns of fragmentation. Exposure to physical shearing creates a characteristic fragment length where DNA fragments are cut in half by shear stress. This characteristic length can be measured using gel electrophoresis or instruments for DNA fragment analysis. Chemical shearing generates randomly distributed fragment lengths visible as a smear of DNA below the peak fragment length. By measuring the peak of DNA fragment length and the proportion of very short DNA fragments both sources of shearing can be measured using commonly used laboratory techniques, providing a suitable quantification of DNA integrity of DNA for sequencing with long-read technologies.

78: CRISPR-Cas12b-assisted nucleic acid detection platform
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Posted 06 Jul 2018

CRISPR-Cas12b-assisted nucleic acid detection platform
3,174 downloads bioRxiv molecular biology

Linxian Li, Shiyuan Li, Jin Wang

Rapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step.

79: Localization microscopy at doubled precision with patterned illumination
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Posted 20 Feb 2019

Localization microscopy at doubled precision with patterned illumination
3,166 downloads bioRxiv molecular biology

Jelmer Cnossen, Taylor Hinsdale, Rasmus Ø. Thorsen, Florian Schueder, Ralf Jungmann, Carlas S. Smith, Bernd Rieger, Sjoerd Stallinga

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field of-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures.

80: In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges
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Posted 26 Jun 2020

In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges
3,159 downloads bioRxiv molecular biology

Beata Turoňová, Mateusz Sikora, Christoph Schürmann, Wim J. H. Hagen, Sonja Welsch, Florian E. C. Blanc, Sören von Bülow, Michael Gecht, Katrin Bagola, Cindy Hörner, Ger van Zandbergen, Shyamal Mosalaganti, Andre Schwarz, Roberto Covino, Michael D. Mühlebach, Gerhard Hummer, Jacomine Krijnse Locker, Martin Beck

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ . Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453. ### Competing Interest Statement The authors have declared no competing interest.

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