scholarly journals A Single Molecule Investigation of I-Motif: Stability, Folding Kinetics, and Potential as an In-situ pH Sensor

2021 ◽  
Author(s):  
Golam Mustafa ◽  
Prabesh Gyawali ◽  
Jacob A. Taylor ◽  
Parastoo Maleki ◽  
Marlon V. Nunez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ph Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Telomeric Sequence ◽  
Folding Kinetics ◽  
Single Molecule Level ◽  
Oxygen Scavenging ◽  
Kinetics Of

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for physiologically low pH levels (pH<6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule F&oumlrster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the kinetics of i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor establish the time scale for the pH drop in a commonly used oxygen scavenging system.

scholarly journals Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene

2021 ◽  
Author(s):  
Guglielmo Vesco ◽  
Marco Lamperti ◽  
Domenico Salerno ◽  
Claudia Adriana Marrano ◽  
Valeria Cassina ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fine Tuning ◽  
Folding Kinetics ◽  
Single Molecule Level ◽  
Double Stranded Dna ◽  
G Quadruplex ◽  
Final Equilibrium State ◽  
Complex Relationships

Abstract G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

scholarly journals Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

2019 ◽  
Vol 116 (10) ◽  
pp. 4275-4284 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Open Source Software ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Level ◽  
Wide Range ◽  
Different Color

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil “stalk” of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

scholarly journals Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET-based Dynamic Nanosensor

2021 ◽  
Author(s):  
Kunal Khanna ◽  
Shankar Mandal ◽  
Aaron T Blanchard ◽  
Muneesh Tewari ◽  
Alexander Johnson-Buck ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Specificity ◽  
Digital Pcr ◽  
Clinical Diagnostics ◽  
Fluorescent Detection ◽  
Single Molecule Level

Biofluids contain cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) that have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that increase the risk of sample loss and cross-contamination, respectively. To achieve the direct, rapid detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein describe an accelerated amplification-free single-molecule kinetic fingerprinting. This approach, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS), exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures individual target molecules onto the surface, while the other transiently interrogates them to generate kinetic fingerprints by intramolecular sin-gle-molecule Forster resonance energy transfer (smFRET). Formamide is used to further accelerate the kinetics of probe-target interactions and fingerprinting, while background signals are reduced by removing non-target-bound probes from the surface using toehold-mediated strand displacement. We show that iSiMREPS can detect in as little as 10 seconds two distinct, promising cancer biomarkers, miR-141 and a common EGFR exon 19 deletion, reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.

Minimalist Approaches to Protein Labelling: Getting the Most Fluorescent Bang for Your Steric Buck

2014 ◽  
Vol 67 (5) ◽  
pp. 686 ◽  
Author(s):  
Lee C. Speight ◽  
Moumita Samanta ◽  
E. James Petersson
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Unnatural Amino Acid ◽  
Spectroscopic Techniques ◽  
Environmental Sensitivity ◽  
Protein Labelling ◽  
Single Molecule Level ◽  
Wavelength Absorption

Fluorescence methods allow one to monitor protein conformational changes, protein–protein associations, and proteolysis in real time, at the single molecule level and in living cells. The information gained in such experiments is a function of the spectroscopic techniques used and the strategic placement of fluorophore labels within the protein structure. There is often a trade-off between size and utility for fluorophores, whereby large size can be disruptive to the protein’s fold or function, but valuable characteristics, such as visible wavelength absorption and emission or brightness, require sizable chromophores. Three major types of fluorophore readouts are commonly used: (1) Förster resonance energy transfer (FRET); (2) photoinduced electron transfer (PET); and (3) environmental sensitivity. This review focuses on those probes small enough to be incorporated into proteins during ribosomal translation, which allows the probes to be placed on the interiors of proteins as they are folded during synthesis. The most broadly useful method for doing so is site-specific unnatural amino acid (UAA) mutagenesis. We discuss the use of UAA probes in applications relying on FRET, PET, and environmental sensitivity. We also briefly review other methods of protein labelling and compare their relative merits to UAA mutagenesis. Finally, we discuss small probes that have thus far been used only in synthetic peptides, but which have unusual value and may be candidates for incorporation using UAA methods.

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