Nanomechanics and co-transcriptional folding of Spinach and Mango

Abstract Recent advances in fluorogen-binding “light-up” RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding in iMangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications.

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Nanomechanics and co-transcriptional folding of Spinach and Mango

10.1101/650119 ◽

2019 ◽

Author(s):

Jaba Mitra ◽

Taekjip Ha

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Mechanical Stability ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Rna Aptamers ◽

Cellular Processes ◽

Force Relaxation ◽

And Function

AbstractRecent advances in fluorogen-binding RNA aptamers known as “light-up” aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn. Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K+, which is needed to stabilize the GQ cores, Mg2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.

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POT1 Stability and Binding Measured by Fluorescence Thermal Shift Assays

10.1101/2021.01.01.425048 ◽

2021 ◽

Author(s):

Lynn W. DeLeeuw ◽

Robert C. Monsen ◽

Vytautas Petrauskas ◽

Robert D. Gray ◽

Lina Baranauskiene ◽

...

Keyword(s):

Thermal Stability ◽

Dna Binding ◽

Competitive Inhibition ◽

Single Stranded Dna ◽

Biophysical Characterization ◽

Drug Candidates ◽

G Quadruplex ◽

And Function ◽

Thermal Shift

AbstractThe protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 – DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

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Single Molecule Fluorescence Resonance Energy Transfer and Ensemble Biophysical Characterization of a G-quadruplex Formed in the Promoter of Human Myocyte Enhancer Factor 2D

Acta Physico-Chimica Sinica ◽

10.3866/pku.whxb20100439 ◽

2010 ◽

Vol 26 (04) ◽

pp. 1099-1106

Author(s):

ZHOU Wen-Hua ◽

◽

YING Li-Ming

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fluorescence ◽

Biophysical Characterization ◽

G Quadruplex ◽

Enhancer Factor

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POT1 stability and binding measured by fluorescence thermal shift assays

PLoS ONE ◽

10.1371/journal.pone.0245675 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0245675

Author(s):

Lynn W. DeLeeuw ◽

Robert C. Monsen ◽

Vytautas Petrauskas ◽

Robert D. Gray ◽

Lina Baranauskiene ◽

...

Keyword(s):

Thermal Stability ◽

Dna Binding ◽

Competitive Inhibition ◽

Single Stranded Dna ◽

Biophysical Characterization ◽

Drug Candidates ◽

G Quadruplex ◽

And Function ◽

Thermal Shift

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 –DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

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Functional characterization of human brown adipose tissue metabolism

Biochemical Journal ◽

10.1042/bcj20190464 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1261-1286 ◽

Cited By ~ 3

Author(s):

Marie Anne Richard ◽

Hannah Pallubinsky ◽

Denis P. Blondin

Keyword(s):

Adipose Tissue ◽

Brown Adipose Tissue ◽

Functional Characterization ◽

Human Infants ◽

Positron Emission ◽

Brown Adipose ◽

Treatment Of Obesity ◽

And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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DNA sliding and loop formation by E. coli SMC complex: MukBEF

10.1101/2021.07.17.452765 ◽

2021 ◽

Author(s):

man zhou

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Dna Interaction ◽

Loop Formation ◽

Unknown Mechanism ◽

E Coli ◽

Dna Compaction ◽

And Function ◽

Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

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Biophysical characterization of DNA binding from single molecule force measurements

Physics of Life Reviews ◽

10.1016/j.plrev.2010.06.001 ◽

2010 ◽

Vol 7 (3) ◽

pp. 299-341 ◽

Cited By ~ 129

Author(s):

Kathy R. Chaurasiya ◽

Thayaparan Paramanathan ◽

Micah J. McCauley ◽

Mark C. Williams

Keyword(s):

Dna Binding ◽

Single Molecule ◽

Force Measurements ◽

Biophysical Characterization

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Biophysical methods toolbox to study ABC exporter structure and function

Biological Chemistry ◽

10.1515/hsz-2016-0244 ◽

2017 ◽

Vol 398 (2) ◽

pp. 229-235

Author(s):

Thomas Marcellino ◽

Vasundara Srinivasan

Keyword(s):

Membrane Proteins ◽

Abc Transporter ◽

Integrated Approach ◽

Structure And Function ◽

Dynamic Membrane ◽

Biophysical Characterization ◽

Intermediate States ◽

And Function

Abstract ABC exporters are highly dynamic membrane proteins that span a huge spectrum of different conformations. A detailed integrated approach of cellular, biochemical and biophysical characterization of these ‘open’, ‘closed’ and other intermediate states is central to understanding their function. Almost 40 years after the discovery of the first ABC transporter, thanks to the enormous development in methodologies, a picture is slowly emerging to visualize how these fascinating molecules transport their substrates. This mini review summarizes some of the biophysical tools that have made a major impact in understanding the function of the ABC exporters.

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