Publications


Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

Scientific Reports Nature Research 9 (2019) 16219

N Robb, J Taylor, A Kent, A Kapanidis, O Pambos, B Gilboa


Coming together during viral assembly.

Nature reviews. Microbiology 16 (2018) 721-721

C Hepp, NC Robb


Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 984-984

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, SR Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.


Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Nature methods 15 (2018) 669-676

B Hellenkamp, S Schmid, O Doroshenko, O Opanasyuk, R Kühnemuth, S Rezaei Adariani, B Ambrose, M Aznauryan, A Barth, V Birkedal, ME Bowen, H Chen, T Cordes, T Eilert, C Fijen, C Gebhardt, M Götz, G Gouridis, E Gratton, T Ha, P Hao, CA Hanke, A Hartmann, J Hendrix, LL Hildebrandt, V Hirschfeld, J Hohlbein, B Hua, CG Hübner, E Kallis, AN Kapanidis, J-Y Kim, G Krainer, DC Lamb, NK Lee, EA Lemke, B Levesque, M Levitus, JJ McCann, N Naredi-Rainer, D Nettels, T Ngo, R Qiu, NC Robb, C Röcker, H Sanabria, M Schlierf, T Schröder, B Schuler, H Seidel, L Streit, J Thurn, P Tinnefeld, S Tyagi, N Vandenberk, AM Vera, KR Weninger, B Wünsch, IS Yanez-Orozco, J Michaelis, CAM Seidel, TD Craggs, T Hugel

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.


Single-molecule analysis of the influenza virus replication initiation mechanism

Biophysical Journal Biophysical Society 114 (2018) 246A-246A

N Robb, AJW te Velthuis, E Fodor, A Kapanidis


Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes

Nucleic Acids Research Oxford University Press 46 (2017) 677-688

D Duchi, K Gryte, NC Robb, Z Morichaud, C Sheppard, K Brodolin, S Wigneshweraraj, A Kapanidis

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.


Preparation of DNA Substrates and Functionalized Glass Surfaces for Correlative Nanomanipulation and Colocalization (NanoCOSM) of Single Molecules.

Methods in enzymology 582 (2017) 275-296

C Duboc, J Fan, ET Graves, TR Strick

Simultaneous nanomanipulation and colocalization of single molecules (NanoCOSM) provides a unique opportunity to correlate the mechanical properties and activities of biomolecules with their conformational states or states of assembly as part of dynamic macromolecular complexes. This opens the door to real-time single-molecule analysis of the correlations between structure, function, and composition of large multicomponent protein complexes.


Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

Nucleic Acids Research Oxford University Press (2016)

NC Robb, AJW te Velthuis, R Wieneke, R Tampe, T Cordes, E Fodor, AN Kapanidis

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provides insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.


RNA polymerase pausing during initial transcription

Molecular cell Cell Press 63 (2016) 939-950

D Duchi, DLV Bauer, L Fernandez, G Evans, N Robb, LC Hwang, K Gryte, A Tomescu, P Zawadzki, Z Morichaud, K Brodolin, A Kapanidis

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.


Reconstruction of bacterial transcription-coupled repair at single-molecule resolution.

Nature 536 (2016) 234-237

J Fan, M Leroux-Coyau, NJ Savery, TR Strick

Escherichia coli Mfd translocase enables transcription-coupled repair by displacing RNA polymerase (RNAP) stalled on a DNA lesion and then coordinating assembly of the UvrAB(C) components at the damage site. Recent studies have shown that after binding to and dislodging stalled RNAP, Mfd remains on the DNA in the form of a stable, slowly translocating complex with evicted RNAP attached. Here we find, using a series of single-molecule assays, that recruitment of UvrA and UvrAB to Mfd-RNAP arrests the translocating complex and causes its dissolution. Correlative single-molecule nanomanipulation and fluorescence measurements show that dissolution of the complex leads to loss of both RNAP and Mfd. Subsequent DNA incision by UvrC is faster than when only UvrAB(C) are available, in part because UvrAB binds 20-200 times more strongly to Mfd–RNAP than to DNA damage. These observations provide a quantitative framework for comparing complementary DNA repair pathways in vivo.


The role of the priming loop in influenza A virus RNA synthesis

Nature Microbiology Nature (2016)

A Te Velthuis, N Robb, AN Kapanidis, E Fodor

RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1 . They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb1,2. Conserved RdRp motifs A–F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation1 . For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism3. The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication4. To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.


The role of the priming loop in influenza A virus RNA synthesis.

Nature microbiology 1 (2016) 16029-

AJW Te Velthuis, NC Robb, AN Kapanidis, E Fodor

RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes(1). They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb(1,2). Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation(1). For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism(3). The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication(4). To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.


DNA replication: Unlocking the secrets of fork arrest.

Nature chemical biology 11 (2015) 550-551

J Fan, TR Strick


A dynamic DNA-repair complex observed by correlative single-molecule nanomanipulation and fluorescence.

Nature structural & molecular biology 22 (2015) 452-457

ET Graves, C Duboc, J Fan, F Stransky, M Leroux-Coyau, TR Strick

We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at ∼4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.


Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter.

Proceedings of the National Academy of Sciences of the United States of America 111 (2014) E3335-E3342

AI Tomescu, NC Robb, N Hengrung, E Fodor, AN Kapanidis

The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction.


Stopped in its tracks: the RNA polymerase molecular motor as a robust sensor of DNA damage.

DNA repair 20 (2014) 49-57

K Howan, J Monnet, J Fan, TR Strick

DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to diffusion of biochemical components and stochasticity of chemical reaction steps. Here we discuss recent experiments using DNA nanomanipulation in a magnetic trap to study the initiation of transcription-coupled repair in a model bacterial system comprising the canonical Escherichia coli RNA polymerase and the Mfd translocase which specifically binds to it. These experiments provide kinetic insight into the reaction process, helping to explain how Mfd discriminates between transcribing RNAP and stalled RNAP. They also identify a reliably long-lived intermediate containing Mfd translocase and, potentially, RNA polymerase. This intermediate presumably serves as a platform for assembly of downstream repair components UvrAB(C).


The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: implications for transcription start-site selection.

J Mol Biol 425 (2013) 875-885

NC Robb, T Cordes, LC Hwang, K Gryte, D Duchi, TD Craggs, Y Santoso, S Weiss, RH Ebright, AN Kapanidis

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RP(o)). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5' end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RP(o). The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RP(o) with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RP(o) using different FRET rulers and labeling positions. An analysis of FRET distributions of RP(o) using burst variance analysis reveals conformational fluctuations in RP(o) in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RP(o). Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RP(o) and indicates that DNA dynamics within the bubble affect the search for transcription start sites.


The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: Implications for transcription start-site selection

Journal of Molecular Biology 425 (2013) 875-885

NC Robb, T Cordes, LC Hwang, K Gryte, D Duchi, TD Craggs, Y Santoso, S Weiss, RH Ebright, AN Kapanidis

Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~ 14 bp around the transcription start site and forms a single-stranded "transcription bubble" within a catalytically active RNAP-DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion ("scrunching") or bubble contraction ("unscrunching"). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RP o using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites. ©2013 Elsevier Ltd. All rights reserved.


Characterizing Influenza a RNA Polymerase - Promoter Interaction using Ensemble Fluorescence Spectroscopy

BIOPHYSICAL JOURNAL 104 (2013) 584A-584A

AI Tomescu, NC Robb, N Hengrung, E Fodor, AN Kapanidis


Characterization of an Ac transposon system based on apt1-m1 (Ac) on the long arm of maize chromosome 9.

Genetica 140 (2012) 337-347

F Wang, P Li, Y Tang, J Fan, D Xu, S Guo, Z Xu, R Song

Activator/Dissociation (Ac/Ds) transposable elements have been used in maize insertional mutagenesis as a complement to Mutator (Mu). In this study, to further improve the efficiency of the Ac/Ds mutagenesis system, we adopted apt1-m1 (Ac) on the long arm of chromosome 9 (9L) as a donor Ac to create an Ac insertion library. This system is based on the negative selection pressure against the donor Ac, and it was highly efficient for isolating new transposition events. We obtained 9,625 transposition events from 1083 F1 ears with an average transposition rate of 8.66 % (rates ranged from 1.11 to 29.73 %). We also adopted a modified PCR-based genome walking strategy to improve the efficiency of the new method for isolating transposon-flanking sequences. This method is more efficient than the Southern-based method that was used in previous studies. A validation step was developed to distinguish transposon tags derived from newly transposed Ac or Ds elements. Using this PCR-based method, we isolated 67 inheritable flanking sequences from the apt1-m1 (Ac) transposition library; of these, 51 were confirmed as tr-Ac-flanking sequences and 11 were tr-Ds-flanking sequences. Similar to other Ac donors from different loci, the apt1-m1 (Ac) system also exhibited a preference for short distance transposition. In this study, we have further improved the Ac mutagenesis system in maize for gene isolation and functional genomics studies.

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