Single Molecule Studies on G-Quadruplex, Protein, and Small Molecule Interactions

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

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Monitoring Small Molecule and G-Quadruplex Interactions and Kinetics using Single Molecule FRET

Biophysical Journal ◽

10.1016/j.bpj.2016.11.829 ◽

2017 ◽

Vol 112 (3) ◽

pp. 151a

Author(s):

Parastoo Maleki

Keyword(s):

Small Molecule ◽

Single Molecule ◽

Single Molecule Fret ◽

G Quadruplex

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Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase

Nucleic Acids Research ◽

10.1093/nar/gkz803 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10744-10753 ◽

Cited By ~ 4

Author(s):

Parastoo Maleki ◽

Golam Mustafa ◽

Prabesh Gyawali ◽

Jagat B Budhathoki ◽

Yue Ma ◽

...

Keyword(s):

Gene Expression ◽

Thermal Stability ◽

Small Molecule ◽

Single Molecule ◽

Telomerase Activity ◽

G Quadruplex ◽

Small Molecule Ligands ◽

The Impact ◽

Different Levels ◽

Thermal Stability Of

Abstract G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.

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Single Molecule Investigations of Protein and Small Molecule Interactions with G-Quadruplexes and their Impact on Telomere Structure

Biophysical Journal ◽

10.1016/j.bpj.2020.11.461 ◽

2021 ◽

Vol 120 (3) ◽

pp. 34a

Author(s):

Hamza Balci ◽

Golam Mustafa ◽

Prabesh Gyawali ◽

Parastoo Maleki ◽

Sajad A. Shiekh

Keyword(s):

Small Molecule ◽

Single Molecule ◽

Molecule Interactions ◽

Telomere Structure

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Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725442653.793522148 ◽

2016 ◽

Author(s):

Olivier Hyrien

Keyword(s):

Single Molecule ◽

Helicase Loading ◽

Origin Licensing ◽

Single Molecule Studies

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Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725442653.793506614 ◽

2015 ◽

Author(s):

Antoine van Oijen

Keyword(s):

Single Molecule ◽

Helicase Loading ◽

Origin Licensing ◽

Single Molecule Studies

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Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

International Journal of Molecular Sciences ◽

10.3390/ijms22052398 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2398

Author(s):

Wooyoung Kang ◽

Seungha Hwang ◽

Jin Young Kang ◽

Changwon Kang ◽

Sungchul Hohng

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Facilitated Diffusion ◽

One Dimensional ◽

Bacterial Transcription ◽

Fluorescence Measurements ◽

Dimensional Diffusion ◽

Single Molecule Studies ◽

Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

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Breaking Down the Supramolecular Ensemble: Single-Molecule Studies of the Concentration Dependence of Main-Chain Supramolecular Polymer Molecular Weight Distributions on Surfaces

Australian Journal of Chemistry ◽

10.1071/ch09615 ◽

2010 ◽

Vol 63 (4) ◽

pp. 624

Author(s):

Michael J. Serpe ◽

Jason R. Whitehead ◽

Stephen L. Craig

Keyword(s):

Molecular Weight ◽

Single Molecule ◽

Monomer Concentration ◽

Supramolecular Polymer ◽

Force Microscopy ◽

Molecular Weight Distributions ◽

Atomic Force ◽

Multi Stage ◽

Single Molecule Studies ◽

Supramolecular Ensemble

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.

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