scholarly journals Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers

2020 ◽  
Author(s):  
Mona Seifert ◽  
Subhas Chandra Bera ◽  
Pauline van Nies ◽  
Robert N. Kirchdoerfer ◽  
Ashleigh Shannon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Therapeutic Strategies ◽  
High Fidelity ◽  
Experimental Paradigm ◽  
Nucleotide Analogs ◽  
The Third ◽  
Nucleotide Addition ◽  
A Chain ◽  
Chain Terminator

SummaryCoronavirus Disease 2019 (COVID-19) results from an infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the third coronavirus outbreak to plague humanity this century. Currently, the most efficacious therapeutic against SARS-CoV-2 infection is the Remdesivir (RDV), an adenine-like ribonucleotide analogue that is very efficiently incorporated by the SARS-CoV-2 replicase. Understanding why RDV is so well incorporated will facilitate development of even more effective therapeutics. Here, we have applied a high-throughput, single-molecule, magnetic-tweezers platform to study thousands of cycles of nucleotide addition by the SARS-CoV-2 replicase in the absence and presence of RDV, a Favipiravir-related analog (T-1106), and the endogenously produced ddhCTP. Our data are consistent with two parallel catalytic pathways of the replicase: a high-fidelity catalytic (HFC) state and a low-fidelity catalytic (LFC) state, the latter allowing the slow incorporation of both cognate and non-cognate nucleotides. ddhCTP accesses HFC, T-1106 accesses LFC as a non-cognate nucleotide, while RDV efficiently accesses both LFC pathway. In contrast to previous reports, we provide unequivocal evidence against RDV functioning as a chain terminator. We show that RDV incorporation transiently stalls the replicase, only appearing as termination events when traditional, gel-based assays are used. The efficiency of ddhCTP utilization by the SARS-CoV-2 replicase suggests suppression of its synthesis during infection, inspiring new therapeutic strategies. Use of this experimental paradigm will be essential to the development of therapeutic nucleotide analogs targeting polymerases.

scholarly journals Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mona Seifert ◽  
Subhas C Bera ◽  
Pauline van Nies ◽  
Robert N Kirchdoerfer ◽  
Ashleigh Shannon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rna Synthesis ◽  
Vaccine Development ◽  
Magnetic Tweezers ◽  
Experimental Paradigm ◽  
Antiviral Protein ◽  
Nucleotide Analogs ◽  
Poor Understanding ◽  
The Impact ◽  
Near Future

The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

scholarly journals Flow Magnetic Tweezers: Imaging Rare Enzymatic Events with Single Molecule Precision

2021 ◽  
Vol 120 (3) ◽  
pp. 33a-34a
Author(s):  
Rohit Agarwal ◽  
Karl E. Duderstadt
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers

scholarly journals Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jason C Bell ◽  
Bian Liu ◽  
Stephen C Kowalczykowski
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Intramolecular Interactions ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Intramolecular Condensation ◽  
Nucleoprotein Complexes ◽  
Ssdna Binding Proteins ◽  
Dna Maintenance

Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.

scholarly journals METAPHASE I ORIENTATION OF CHAIN-FORMING INTERCHANGE QUADRIVALENTS: A THEORETICAL CONSIDERATION

Genetics ◽  
1984 ◽  
Vol 108 (3) ◽  
pp. 707-718
Author(s):  
Prasad R K Koduru
Keyword(s):  
Pearl Millet ◽  
Theoretical Consideration ◽  
Pennisetum Americanum ◽  
Orientation Behavior ◽  
The Third ◽  
A Chain ◽  
Metaphase I

ABSTRACT The orientation behavior of chain forming interchange quadrivalents at metaphase I was studied in three interchange heterozygotes of pearl millet [Pennisetum americanum (L.) Leeke] which involve chromosomes 1, 3, 6 and 7 in various combinations. Of these, two combinations predominantly produced rings and the third was a chain-forming type. The chain quadrivalents derived from the two ring-forming interchanges, as well as the chain quadrivalent generated by the third interchange, all showed one adjacent orientation at metaphase I (adjacent-1 or -2, depending upon the formation or failure of chiasmata and their positions in the different segments of the pachytene cross). Homologous centromere co-orientation leading to adjacent-1 and alternate-1 occurs following chiasma failure in the noncentric arms of the pachytene cross, and nonhomologous centromere co-orientation leading to adjacent-2 and alternate-2 occurs following chiasma failure in the centric arms of the pachytene cross. Thus, it has been proposed that, unlike in ring quadrivalents, a specific chain quadrivalent will have only homologous or nonhomologous centromere co-orientations at metaphase I.

scholarly journals Closing and opening of the RNA polymerase trigger loop

2019 ◽  
Author(s):  
Abhishek Mazumder ◽  
Miaoxin Lin ◽  
Achillefs N. Kapanidis ◽  
Richard H. Ebright
Keyword(s):  
Rna Polymerase ◽  
Fluorescent Probe ◽  
Active Center ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fluorescence ◽  
Structural Element ◽  
Trigger Loop ◽  
Nucleotide Addition

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an “unfolded”/“open” state that allows an NTP substrate to enter the active center and a “folded”/“closed” state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

scholarly journals Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

2021 ◽  
Vol 118 (33) ◽  
pp. e2025578118
Author(s):  
Lena Voith von Voithenberg ◽  
Anders Barth ◽  
Vanessa Trauschke ◽  
Benjamin Demarco ◽  
Swati Tyagi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational Space ◽  
Distribution Analysis ◽  
Nucleotide Exchange ◽  
Nucleotide Analogs ◽  
Recognition Mechanism ◽  
Substrate Peptide

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

A spectroscopic case for SPSi detection: The third-row in a single molecule

2016 ◽  
Vol 145 (12) ◽  
pp. 124311 ◽  
Author(s):  
Brian Finney ◽  
Ryan C. Fortenberry ◽  
Joseph S. Francisco ◽  
Kirk A. Peterson
Keyword(s):  
Single Molecule ◽  
The Third

scholarly journals Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

2021 ◽  
Author(s):  
Terren Chang ◽  
Xi Long ◽  
Shankar Shastry ◽  
Joseph William Parks ◽  
Michael D Stone
Keyword(s):  
Single Molecule ◽  
Mechanical Analysis ◽  
Magnetic Tweezers ◽  
Repeat Sequence ◽  
Single Stranded Dna ◽  
Telomere Repeat ◽  
D Loop ◽  
Human Telomere ◽  
Strand Invasion

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

Magnetic Tweezers-Based Single-Molecule Assays to Study Interaction of E. coli SSB with DNA and

2021 ◽  
pp. 93-115
Author(s):  
Debjani Bagchi ◽  
Weiting Zhang ◽  
Samar Hodeib ◽  
Bertrand Ducos ◽  
Vincent Croquette ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
E Coli
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