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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 83,713 bioRxiv papers from 360,555 authors.

Most downloaded bioRxiv papers, since beginning of last month

in category molecular biology

2,724 results found. For more information, click each entry to expand.

1: LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space
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Posted to bioRxiv 08 Apr 2020

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space
10,669 downloads molecular biology

Jonathan L. Schmid-Burgk, David Li, David Feldman, Mikołaj Słabicki, Jacob Borrajo, Jonathan Strecker, Brian Cleary, Aviv Regev, Feng Zhang

The ongoing COVID-19 pandemic has already caused devastating losses. Early evidence shows that the exponential spread of COVID-19 can be slowed by restrictive isolation measures, but these place a tremendous burden on society. Moreover, once these restrictions are lifted, the exponential spread is likely to re-emerge. It has been suggested that population-scale testing can help break the cycle of isolation and spread, but current detection methods are not capable of such large-scale processing. Here we propose LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) protocol that could dramatically reduce the cost and complexity of population-scale testing. In this approach, individual samples are processed in a single heat step, producing barcoded amplicons that can be shipped to a sequencing center, pooled, and analyzed en masse. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, significantly reducing cost and organizational efforts. Given the low cost and scalability of next-generation sequencing, we believe that this method can be affordably scaled to analyze millions of samples per day using existing sequencing infrastructure.

2: Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
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Posted to bioRxiv 20 Apr 2020

Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
8,986 downloads molecular biology

Daniel J Butler, Christopher Mozsary, Cem Meydan, David Danko, Jonathan Foox, Joel Rosiene, Alon Shaiber, Ebrahim Afshinnekoo, Matthew MacKay, Fritz J. Sedlazeck, Nikolay A Ivanov, Maria Sierra, Diana Pohle, Michael Zietz, Undina Gisladottir, Vijendra Ramlall, Craig D Westover, Krista Ryon, Benjamin Young, Chandrima Bhattacharya, Phyllis Ruggiero, Bradley W. Langhorst, Nathan Tanner, Justyna Gawrys, Dmitry Meleshko, Dong Xu, Peter A D Steel, Amos J Shemesh, Jenny Xiang, Jean Thierry-Mieg, Danielle Thierry-Mieg, Robert E. Schwartz, Angelika Iftner, Daniela Bezdan, John Sipley, Lin Cong, Arryn Craney, Priya Velu, Ari M. Melnick, Iman Hajirasouliha, Stacy M. Horner, Thomas Iftner, Mirella Salvatore, Massimo Loda, Lars F Westblade, Melissa Cushing, Shawn Levy, Shixiu Wu, Nicholas P. Tatonetti, Marcin Imielinski, Hanna Rennert, Christopher E. Mason

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in ACE, interferon, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health monitoring, and new therapeutic targets. ### Competing Interest Statement N.T. and B.L. are employees at New England Biolabs.

3: The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells
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Posted to bioRxiv 31 Jan 2020

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells
8,082 downloads molecular biology

Markus Hoffmann, Hannah Kleine-Weber, Nadine Krüger, Marcel Müller, Christian Drosten, Stefan Pöhlmann

The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum from a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention.

4: Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir
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Posted to bioRxiv 09 Apr 2020

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir
6,262 downloads molecular biology

Wanchao Yin, Chunyou Mao, Xiaodong Luan, Dan-Dan Shen, Qingya Shen, Haixia Su, Xiaoxi Wang, Fulai Zhou, Wenfeng Zhao, Minqi Gao, Shenghai Chang, Yuan-Chao Xie, Guanghui Tian, He-Wei Jiang, Sheng-Ce Tao, Jingshan Shen, Yi Jiang, Hualiang Jiang, H. Eric Xu, Shuyang Zhang, Yan Zhang, H. Eric Xu

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 Å. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection. ### Competing Interest Statement The authors have declared no competing interest.

5: An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12
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Posted to bioRxiv 02 Mar 2020

An ultrasensitive, rapid, and portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12
4,722 downloads molecular biology

Curti Lucia, Pereyra-Bonnet Federico, Gimenez Carla Alejandra

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received global attention due to the recent outbreak in China. In this work, we report a CRISPR-Cas12 based diagnostic tool to detect synthetic SARS-CoV-2 RNA sequences in a proof-of-principle evaluation. The test proved to be sensitive, rapid, and potentially portable. These key traits of the CRISPR method are critical for virus detection in regions that lack resources to use the currently available methods.

6: Preliminary support for a 'dry swab, extraction free' protocol for SARS-CoV-2 testing via RT-qPCR
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Posted to bioRxiv 23 Apr 2020

Preliminary support for a 'dry swab, extraction free' protocol for SARS-CoV-2 testing via RT-qPCR
3,687 downloads molecular biology

Sanjay Srivatsan, Peter D. Han, Katrina van Raay, Caitlin R Wolf, Denise J. McCulloch, Ashley E Kim, Elisabeth Brandstetter, Beth Martin, Jase Gehring, Wei Chen, Seattle Flu Study Investigators, Sriram Kosuri, Eric Q. Konnick, Christina M. Lockwood, Mark J. Rieder, Deborah A. Nickerson, Helen Y. Chu, Jay Shendure, Lea M Starita

The urgent need for massively scaled clinical or surveillance testing for SARS-CoV-2 has necessitated a reconsideration of the methods by which respiratory samples are collected, transported, processed and tested. Conventional testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab, storage of the swab during transport in universal transport medium (UTM), extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). As testing has scaled across the world, supply chain challenges have emerged across this entire workflow. Here we sought to evaluate how eliminating the UTM storage and RNA extraction steps would impact the results of molecular testing. Using paired mid-turbinate swabs self-collected by 11 individuals with previously established SARS-CoV-2 positivity, we performed a comparison of conventional (swab → UTM → RNA extraction → RT-qPCR) vs. simplified (direct elution from dry swab → RT-qPCR) protocols. Our results suggest that dry swabs eluted directly into a simple buffered solution (TE) can support molecular detection of SARS-CoV-2 via endpoint RT-qPCR without substantially compromising sensitivity. Although further confirmation with a larger sample size and variation of other parameters is necessary, these results are encouraging for the possibility of a simplified workflow that could support massively scaled testing for COVID-19 control. ### Competing Interest Statement The authors have declared no competing interest.

7: Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites
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Posted to bioRxiv 07 Mar 2020

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites
2,944 downloads molecular biology

Sisi Kang, Mei Yang, Zhongsi Hong, Liping Zhang, Zhaoxia Huang, Xiaoxue Chen, Suhua He, Ziliang Zhou, Zhechong Zhou, Qiuyue Chen, Yan Yan, Changsheng Zhang, Hong Shan, Shoudeng Chen

The outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

8: The SARS-CoV-2 exerts a distinctive strategy for interacting with the ACE2 human receptor
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Posted to bioRxiv 12 Mar 2020

The SARS-CoV-2 exerts a distinctive strategy for interacting with the ACE2 human receptor
2,621 downloads molecular biology

Esther S Brielle, Dina Schneidman-Duhovny, Michal Linial

The COVID-19 disease has plagued over 110 countries and has resulted in over 4,000 deaths within 10 weeks. We compare the interaction between the human ACE2 receptor and the SARS-CoV-2 spike protein with that of other pathogenic coronaviruses using molecular dynamics simulations. SARS-CoV, SARS-CoV-2, and HCoV-NL63 recognize ACE2 as the natural receptor but present a distinct binding interface to ACE2 and a different network of residue-residue contacts. SARS-CoV and SARS-CoV-2 have comparable binding affinities achieved by balancing energetics and dynamics. The SARS-CoV-2 - ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to SARS-CoV. These findings expose an exceptional evolutionary exploration exerted by coronaviruses toward host recognition. We postulate that the versatility of cell receptor binding strategies has immediate implications on therapeutic strategies.

9: Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is an Inconvenient Target for Small-Molecule Inhibitors Design
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Posted to bioRxiv 02 Mar 2020

Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is an Inconvenient Target for Small-Molecule Inhibitors Design
2,230 downloads molecular biology

Maria Bzówka, Karolina Mitusińska, Agata Raczyńska, Aleksandra Samol, Jack A. Tuszynski, Artur Góra

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mix-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar SARS Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins show major differences in both shape and size indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site’s conformational changes during the simulation time indicates its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicates that the virus’ mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design. * Mpro : Main protease CoVs : Coronaviruses ORFs : Open reading frames 3CLpro : Chymotrypsin-like cysteine protease S : Spike surface glycoprotein E : Small envelope protein M : Matrix protein N : Nucleocapsid protein N3 : N-[(5 methylisoxazol-3-yl)carbonyl] alanyl-L-valyl-N~1-((1R,2Z)-4-(benzyloxy)-4-oxo-1--{[(3R)-2-oxopyrrolidin-3-yl]methyl}but-2-enyl)-L-leucinamide cMD : Classical molecular dynamics simulations MixMD : Mixed-solvent molecular dynamics simulations PDB : Protein Data Bank MAV : Maximal accessible volume ACN : Acetonitrile BNZ : Benzene DMSO : Dimethylsulfoxide MEO : Methanol PHN : Phenol URE : Urea CMA : Correlated mutation analysis

10: Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2
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Posted to bioRxiv 03 Mar 2020

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2
2,163 downloads molecular biology

Youngchang Kim, Robert Jedrzejczak, Natalia I. Maltseva, Michael Endres, Adam Godzik, Karolina Michalska, Andrzej Joachimiak

Severe Acute Respiratory Syndrome Coronavirus 2 is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS- and MERS-CoVs the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report the high-resolution crystal structure of endoribonuclease Nsp15/NendoU from SARS-CoV-2 - a virus causing current world-wide epidemics. We compare this structure with previously reported models of Nsp15 from SARS and MERS coronaviruses.

11: Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic
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Posted to bioRxiv 08 Apr 2020

Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic
2,097 downloads molecular biology

Paul R. Grant, Melanie A Turner, Gee Yen Shin, Eleni Nastouli, Lisa J Levett

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19), a respiratory tract infection. The standard molecular diagnostic test is a multistep process involving viral RNA extraction and real-time quantitative reverse transcriptase PCR (qRT-PCR). Laboratories across the globe face constraints on equipment and reagents during the COVID-19 pandemic. We have developed a simplified qRT-PCR assay that removes the need for an RNA extraction process and can be run on a real-time thermal cycler. The assay uses custom primers and probes, and maintains diagnostic sensitivity within 98.0% compared to the assay run on a high-throughput, random-access automated platform, the Panther Fusion (Hologic). This assay can be used to increase capacity for COVID-19 testing for national programmes worldwide. ### Competing Interest Statement The authors have declared no competing interest.

12: A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material
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Posted to bioRxiv 21 Apr 2020

A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material
2,093 downloads molecular biology

Jennifer N. Rauch, Eric Valois, Sabrina C Solley, Friederike Braig, Ryan S Lach, Naomi J Baxter, Kenneth S Kosik, Carolina Arias, Diego Acosta-Alvear, Maxwell Z. Wilson

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly-trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance. ### Competing Interest Statement The authors have declared no competing interest.

13: Rapid, field-deployable nucleobase detection and identification using FnCas9
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Posted to bioRxiv 09 Apr 2020

Rapid, field-deployable nucleobase detection and identification using FnCas9
2,012 downloads molecular biology

Mohd. Azhar, Rhythm Phutela, Asgar Hussain Ansari, Dipanjali Sinha, Namrata Sharma, Manoj Kumar, Meghali Aich, Saumya Sharma, Riya Rauthan, Khushboo Singhal, Harsha Lad, Pradeep Kumar Patra, Govind Makharia, Giriraj Ratan Chandak, Debojyoti Chakraborty, Souvik Maiti

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids have provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19. ### Competing Interest Statement D.C., S.M., M.A., R.P., A.S.A, D.S., N.S. and S.S. are authors in provisional patent applications that have been filed in relation to this work.

14: SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction
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Posted to bioRxiv 30 Mar 2020

SARS-CoV-2 detection from nasopharyngeal swab samples without RNA extraction
1,954 downloads molecular biology

Carolina Beltrán-Pavez, Chantal L. Márquez, Gabriela Muñoz, Fernando Valiente-Echeverría, Aldo Gaggero, Ricardo Soto-Rifo, Gonzalo P. Barriga

The ongoing COVID-19 pandemic has reached more than 200 countries and territories worldwide. Given the large requirement of SARS-CoV-2 diagnosis and considering that RNA extraction kits are in short supply, we investigated whether two commercial RT-qPCR kits were compatible with direct SARS-CoV-2 detection from nasopharyngeal swab samples. We show that one of the tested kits is fully compatible with direct SARS-CoV-2 detection suggesting that omission of RNA extraction step should be considered in SARS-CoV-2 diagnosis.

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

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro
1,942 downloads 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, Hualiang 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.

16: A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
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Posted to bioRxiv 31 Mar 2020

A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production
1,941 downloads molecular biology

Sanchita Bhadra, Andre C Maranhao, Andrew D. Ellington

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed a RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase / DNA polymerase (RTX)[1][1]. Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs from Addgene or our lab and begin to develop their own assays, within whatever regulatory framework exists for them. We lay out protocols for dye-based and TaqMan probe-based assays, in order to best compare with ‘gold standard’ reagents. These protocols should form the basis of further modifications that can simplify the assay to the use of overexpressing cells themselves as reagents. Developing dye-based and TaqMan probe-based RT-qPCR assays with RTX ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1

17: A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2
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Posted to bioRxiv 27 Feb 2020

A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2
1,750 downloads molecular biology

Zhen Zhao, Haodong Cui, Wenxing Song, Xiaoling Ru, Wenhua Zhou, Xuefeng Yu

The ongoing outbreak of the novel coronavirus disease 2019 (COVID-19) originating from Wuhan, China, draws worldwide concerns due to its long incubation period and strong infectivity. Although RT-PCR-based molecular diagnosis techniques are being widely applied for clinical diagnosis currently, timely and accurate diagnosis are still limited due to labour intensive and time-consuming operations of these techniques. To address the issue, herein we report the synthesis of poly (amino ester) with carboxyl groups (PC)-coated magnetic nanoparticles (pcMNPs), and the development of pcMNPs-based viral RNA extraction method for the sensitive detection of COVID-19 causing virus, the SARS-CoV-2. This method combines the lysis and binding steps into one step, and the pcMNPs-RNA complexes can be directly introduced into subsequent RT-PCR reactions. The simplified process can purify viral RNA from multiple samples within 20 min using a simple manual method or an automated high-throughput approach. By identifying two different regions (ORFlab and N gene) of viral RNA, a 10-copy sensitivity and a strong linear correlation between 10 and 100,000 copies of SARS-CoV-2 pseudovirus particles are achieved. Benefitting from the simplicity and excellent performances, this new extraction method can dramatically reduce the turn-around time and operational requirements in current molecular diagnosis of COVID-19, in particular for the early clinical diagnosis.

18: One-step RNA extraction for RT-qPCR detection of 2019-nCoV
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Posted to bioRxiv 05 Apr 2020

One-step RNA extraction for RT-qPCR detection of 2019-nCoV
1,736 downloads molecular biology

Monica Sentmanat, Evguenia Kouranova, Xiaoxia Cui

The global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three months[1][1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from three manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher senitivity and improved saftey. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1

19: Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9
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Posted to bioRxiv 30 Mar 2020

Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9
1,571 downloads molecular biology

D. R. Littler, B. S. Gully, R. N. Colson, J Rossjohn

Many of the proteins produced by SARS-CoV-2 have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate both viral replication and virulence. Current understanding suggests that Nsp9 is involved in viral genomic RNA reproduction. Nsp9 is thought to bind RNA via a fold that is unique to this class of betacoronoaviruses although the molecular basis for this remains ill-defined. We sought to better characterise the SARS-CoV-2 Nsp9 protein and subsequently solved its X-ray crystal structure, in an apo-form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The structure of the SARS-CoV-2 Nsp9 revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted on the relative juxtaposition of the monomers within the homodimer. Together we have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure and identified a peptide-binding site that may warrant further study from the perspective of understanding Nsp9 function.

20: A Structure-Based Mechanism for DNA Entry into the Cohesin Ring
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Posted to bioRxiv 21 Apr 2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring
1,540 downloads molecular biology

Torahiko L. Higashi, Patrik Eickhoff, Joana S. Simoes, Julia Locke, Andrea Nans, Helen R. Flynn, Ambrosius P. Snijders, George Papageorgiou, Nicola O’Reilly, Zhuo A Chen, Francis J O'Reilly, Juri Rappsilber, Alessandro Costa, Frank Uhlmann

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA. ### Competing Interest Statement The authors have declared no competing interest.

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