Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon; Panzeri, Francesco; Gulinatti, Angelo; Rech, Ivan; Ghioni, M.; Weiss, Shimon; Michalet, Xavier

doi:10.1101/085027

Cited by 7 publications

(56 citation statements)

References 77 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is important to mention that the kinetics of transcription starting from RP ITC D 2 , measured by the single round quenched kinetics assay, 2 was very similar to the promoter escape kinetics measured by Ingargiola et al with an 8£1 multispot smFRET setup, following the open bubble subpopulation in (¡8)TA¡(¡5)NTD after addition of all NTPs. 59 This indicates that promoter escape kinetics of RP ITC D 2 with dyes in the bubble region follow similar run-off kinetics for unlabeled DNA.…”

Section: Experimental Evidence Of Pausing During Transcription Initiamentioning

confidence: 81%

Different types of pausing modes during transcription initiation

et al. 2017

Self Cite

View full text Add to dashboard Cite

In many cases, initiation is rate limiting to transcription. This due in part to the multiple cycles of abortive transcription that delay promoter escape and the transition from initiation to elongation. Pausing of transcription in initiation can further delay promoter escape. The previously hypothesized pausing in initiation was confirmed by two recent studies from Duchi et al. 1 and from Lerner, Chung et al. 2 In both studies, pausing is attributed to a lack of forward translocation of the nascent transcript during initiation. However, the two works report on different pausing mechanisms. Duchi et al. report on pausing that occurs during initiation predominantly on-pathway of transcript synthesis. Lerner, Chung et al. report on pausing during initiation as a result of RNAP backtracking, which is off-pathway to transcript synthesis. Here, we discuss these studies, together with additional experimental results from single-molecule FRET focusing on a specific distance within the transcription bubble. We show that the results of these studies are complementary to each other and are consistent with a model involving two types of pauses in initiation: a short-lived pause that occurs in the translocation of a 6-mer nascent transcript and a long-lived pause that occurs as a result of 1-2 nucleotide backtracking of a 7-mer transcript. Prolonged periods during which RNA polymerase (RNAP) resides in a specific state can be used for efficient control and regulation of DNA transcription. This drives us to study the slow processes that limit the overall rate of transcription.Transcription is generally divided into three major stages: initiation, elongation and termination, where initiation is usually the rate-limiting stage. Initiation can be further divided into sub-stages.3,4 First, the RNA polymerase (RNAP) holoenzyme is formed by the association of the core enzyme (subunit composition a 2 ,b,b 0 ,v) with the specificity subunit s. Different s factors recognize specific promoter sequences of various groups of genes. The major s factor in Escherichia coli, s70, is responsible for recognition of promoters for house-keeping genes. After association with the RNAP holoenzyme, s70 binds to the promoter DNA to form the RNAP promoter closed complex (RPc), which involves binding at two hexamer sequences centered around positions ¡10 and ¡35 relative to the C1 transcription start site. After binding to the promoter, RNAP forms the RNAP promoter open complex (RPo) through successive isomerization steps. 5,6 During this process around 13 bp of DNA (from registers ¡11 to C2) must melt to form the transcription bubble. The sequence of the template strand of the transcription bubble serves as a reading guide for the polymerization of an RNA transcript through base complementation. The two most downstream DNA bases in the template strand of the transcription bubble are coordinated with the active site of RNAP, where the addition of NTPs into the 3 0 end of the nascent transcript occurs. The downstream fork of the transcription b...

show abstract

Section: Experimental Evidence Of Pausing During Transcription Initiamentioning

confidence: 81%

Different types of pausing modes during transcription initiation

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…The observation volume of confocal microscopy is diffraction-limited and thus it is inherently low throughput. Ingargiola et al used parallelized beams and a single-photon avalanche diode array, and as a result they increased the throughput by 8 ( Figure 1e; Ingargiola et al, 2017). This approach is more suitable to study freely diffusing single-molecules and reveal their fast dynamics.…”

Section: Single-molecule Fret and Beyondmentioning

confidence: 99%

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

Croop

Zhang

Lim

et al. 2019

WIREs Mechanisms of Disease

View full text Add to dashboard Cite

Recent advances in single‐molecule techniques have led to new discoveries in analytical chemistry, biophysics, and medicine. Understanding the structure and behavior of single biomolecules provides a wealth of information compared to studying large ensembles. However, developing single‐molecule techniques is challenging and requires advances in optics, engineering, biology, and chemistry. In this paper, we will review the state of the art in single‐molecule applications with a focus over the last few years of development. The advancements covered will mainly include light‐based in vitro methods, and we will discuss the fundamentals of each with a focus on the platforms themselves. We will also summarize their limitations and current and future applications to the wider biological and chemical fields. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods

show abstract

“…This requires careful magnification adjustments, and cumbersome alignment steps, including adjusting a rotational degree of freedom. For these reasons, we chose a more flexible (if more expensive) solution based on programmable liquid crystal on silicon (LCOS) spatial light modulator (SLM) [27,28,29,30]. These devices can be used in direct space [31] or reciprocal space [32], as used in holography.…”

Section: Setup Descriptionmentioning

confidence: 99%

“…1A) employed a single CW laser and was used to demonstrate high-throughput fluorescence correlation spectroscopy (HT-FCS) and single-molecule detection [27]. We later added a second linear 8-SPAD array to the setup to enable two-color, 8-spot smFRET measurements [29]. Both setups used a 532 nm highpower 68 MHz pulsed laser for historical reasons, although we could not take advantage of the 8 ps pulsed laser excitation with these SPAD arrays.…”

Section: Setup Descriptionmentioning

confidence: 99%

See 1 more Smart Citation

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Segal

Ingargiola

Lerner

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra-and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly-and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.• The spot pitch in the sample plane (5.4 μm) is defined by the pitch between adjacent SPADs (500 μm) divided by the emission path magnification (∼ 90×).• After demagnification by the excitation path, this translates into a 463 μm lenslet pitch on the LCOS, or 23.1 LCOS pixels. During alignment the pitch is optimized to account for the actual excitation path demagnification, by adjusting the pattern by fractions of LCOS 6

show abstract

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

Cited by 7 publications

References 77 publications

Different types of pausing modes during transcription initiation

Different types of pausing modes during transcription initiation

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

Contact Info

Product

Resources

About