Biology subjects

Kapanidis, A.

Publications and source records attributed to Kapanidis, A..

9 recordsLinked to original sources

bioRxiv2025

Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence.

biophysics↗

bioRxiv2025

Tunable fluorogenic DNA probes drive fast and high-resolution single-molecule fluorescence imaging

A main limitation of single-molecule fluorescence (SMF) measurements is the "high concentration barrier", describing the maximum concentration of fluorescent species tolerable for sufficient signal-to-noise ratio (SNR). To address this barrier in several SMF applications, we design fluorogenic probes based on short ssDNAs, fluorescing only upon hybridising to their complementary target sequence. We engineer the quenching efficiency and fluorescence enhancement upon duplex formation through screening several fluorophore-quencher combinations, label lengths, and sequence motifs, which we utilise as tuning screws to adapt our labels to different experimental designs. Using these fluorogenic probes, we can perform SMF experiments at concentrations of 10 {micro}M fluorescent labels; this concentration is 100-fold higher than the operational limit for standard TIRF experiments. We demonstrate the ease of implementing these probes into existing protocols by performing super-resolution imaging with DNA-PAINT, employing a fluorogenic 6 nt-long imager; through the faster acquisition of binding events, the imaging of viral genome segments could be sped up significantly to achieve extraction of 20-nm structural features with only [~]150 s of imaging. The exceptional tunability of our probe design will overcome concentration barriers in SMF experiments and unlock new possibilities in super-resolution imaging, molecular tracking, and smFRET. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/634148v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d51a20org.highwire.dtl.DTLVardef@1a64fb7org.highwire.dtl.DTLVardef@1bbb4bforg.highwire.dtl.DTLVardef@578c63_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

The displacement of the σ70 finger in initial transcription is highly heterogeneous and promoter-dependent

Bacterial sigma factors ({sigma}) contain a highly conserved structural module, the {sigma}-finger, which forms a loop that protrudes towards the RNA polymerase (RNAP) active-centre in the open complex and has been implicated in pre-organisation of template DNA, abortive initiation of short RNAs, initiation pausing, and promoter escape. Here, we introduce a novel single-molecule FRET (smFRET) assay to monitor {sigma}-finger motions during transcription initiation and promoter escape. We find that the {sigma}-finger is displaced from its position inside the active site cleft before promoter escape, and after synthesis of RNAs with lengths that are highly dependent on the sequence of the promoter used. Real-time smFRET measurements reveal the presence of significant heterogeneity in the timing of finger displacement and show that different {sigma}-finger conformations in single open transcription complexes are associated with substantially different kinetics in transcription initiation and promoter escape, potentially impacting gene regulation in bacteria.

biophysics↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2022

Bleaching-resistant single-molecule fluorescence and FRET monitoring based on fluorophore exchange via transient DNA binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Forster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach should enable capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

biophysics↗

bioRxiv2021

Transcription initiation at a consensus bacterial promoter proceeds via a "bind-unwind-load-and-lock" mechanism

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, "bind-unwind-load-and-lock," model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters. Significance statementTranscription initiation, the first step and most important step in gene expression for all organisms, involves unwinding of promoter DNA by RNA polymerase (RNAP) to form an open complex (RPo); this step also underpins transcriptional regulation and serves as an antibiotic target. Despite decades of research, the mechanism of promoter DNA unwinding has remained unresolved. Here, we solve this puzzle by using single-molecule fluorescence to directly monitor conformational changes in the promoter DNA and RNAP in real time during RPo formation. We show that RPo forms via a "bind-unwind-load-and-lock" mechanism, where the promoter unwinds outside the RNAP cleft, the unwound template DNA loads into the cleft, and RNAP "locks" the template DNA in place by closing the RNAP clamp module.

biochemistry↗

bioRxiv2019

Closing and opening of the RNA polymerase trigger loop

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.

molecular biology↗

bioRxiv2018

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp

RNA polymerase (RNAP) contains a mobile structural module, the \"clamp,\" that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including loading of promoter DNA into the RNAP active-center cleft, unwinding of promoter DNA, transcription elongation complex stability, transcription pausing, and transcription termination. Crystal structures and single-molecule FRET studies establish that the clamp can adopt open and closed conformational states; however, the occurrence, pathway, and kinetics of transitions between clamp states have been unclear. Using single-molecule FRET (smFRET) on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme exists in three distinct conformational states: the previously defined open state, the previously defined closed state, and a previously undefined partly closed state. smFRET time-traces show dynamic transitions between open, partly closed, and closed states on the 0.1-1 second time-scale. Similar analyses of transcription initiation complexes confirm that the RNAP clamp is closed in the catalytically competent transcription initiation complex and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, in contrast to in RNAP holoenzyme, the clamp does not interconvert between the closed state and other states. The stringent-response alarmone ppGpp selectively stabilizes the partly-closed-clamp state, inhibiting interconversion between the partly closed state and the open state. The methods of this report should allow elucidation of clamp conformation and dynamics during all phases of transcription.\n\nSIGNIFICANCE STATEMENTThe clamp forms a pincer of the RNA polymerase \"crab-claw\" structure, and adopts many conformations with poorly understood function and dynamics. By measuring distances within single surface-attached molecules, we observe directly the motions of the clamp and show that it adopts an open, a closed, and a partly closed state; the last state is stabilized by a sensor of bacterial starvation, linking the clamp conformation to the mechanisms used by bacteria to counteract stress. We also show that the clamp remains closed in many transcription steps, as well as in the presence of a specific antibiotic. Our approach can monitor clamp motions throughout transcription and offers insight on how antibiotics can stop pathogens by blocking their RNA polymerase movements.

biophysics↗

bioRxiv2017

Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

Fidaxomicin is an antibacterial drug in clinical use in treatment of Clostridium difficile diarrhea1-2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1-4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1-2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6-8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9 and inhibits transcription initiation at an earlier step than Rif8-11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12-13 structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5 [A] resolution. The structure shows that Lpm binds at the base of the RNAP \"clamp,\" interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17{degrees} relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14 experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor {sigma} regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

molecular biology↗