Effect of glycerol on free DNA: A molecular dynamics simulation study

Diaz, Aathithya; Jothiraman, Hari Balaji; Ramakrishnan, Vigneshwar

doi:10.1016/j.jmgm.2022.108169

Cited by 3 publications

(2 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Dwell times start at 1.75 s (state 1) and 2.69 s (state 2) for pure imaging buffer and decrease down to 0.62 s and 0.85 s in buffer containing 30 % (v/v) glycerol. The multi-fold increase in binding kinetics can be explained by a reported destabilization of base-pairing due to changes in the ssDNA hydration shell 35 and concomitantly disturbed hydrogen bonding due to the osmolyte-DNA interaction. The melting enthalpy and melting temperature decreases linearly with glycerol concentration at about 0.2 °C per % (v/v) 36,37 in line with our observations (Figure 5b).…”

Section: Resultsmentioning

confidence: 99%

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

Wanninger

Asadiatouei

Bohlen

et al. 2023

Preprint

View full text Add to dashboard Cite

Single-molecule experiments have changed the way we investigate the physical world but data analysis is typically time-consuming and prone to human bias. Here, we present Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software package consisting of an ensemble of deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, in particular from single-molecule Foerster Resonance Energy Transfer (FRET) experiments. Deep-LASI automatically sorts single molecule traces, determines FRET correction factors and classifies the state transitions of dynamic traces, all in ~20-100 ms per trajectory. We thoroughly benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.

show abstract

Section: Resultsmentioning

confidence: 99%

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

Wanninger

Asadiatouei

Bohlen

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Dwell times start at 1.75 s (state 1) and 2.69 s (state 2) for pure imaging buffer and decrease down to 0.62 s and 0.85 s in buffer containing 30% (v/v) glycerol. The multi-fold increase in binding kinetics can be explained by a reported destabilization of base-pairing due to changes in the ssDNA hydration shell 37 and concomitantly disturbed hydrogen bonding due to the osmolyte-DNA interaction. The melting enthalpy and melting temperature decreases linearly with glycerol concentration at about 0.2 °C per % (v/v) 38 , 39 in line with our observations (Fig.…”

Section: Resultsmentioning

confidence: 99%

Deep-LASI: deep-learning assisted, single-molecule imaging analysis of multi-color DNA origami structures

Wanninger,

Asadiatouei,

Bohlen

et al. 2023

Nat Commun

View full text Add to dashboard Cite

Single-molecule experiments have changed the way we explore the physical world, yet data analysis remains time-consuming and prone to human bias. Here, we introduce Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite powered by deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, especially from single-molecule Förster Resonance Energy Transfer (smFRET) experiments. Deep-LASI automatically sorts recorded traces, determines FRET correction factors and classifies the state transitions of dynamic traces all in ~20–100 ms per trajectory. We benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.

show abstract

Molecular Crowding Suppresses Mechanical Stress-Driven DNA Strand Separation

Desai,

Marko

2024

Preprint

View full text Add to dashboard Cite

Molecular crowding influences DNA mechanics and DNA - protein interactions and is ubiquitous in living cells. Quantifying the effects of molecular crowding on DNA supercoiling is essential to relatingin-vitroexperiments toin-vivoDNA supercoiling. We use single molecule magnetic tweezers to study DNA supercoiling in the presence of dehydrating or crowding co-solutes. To study DNA supercoiling, we apply a stretching force of 0.8 pN to the DNA and then rotate one end of the DNA to induce supercoiling. In a 200 mM NaCl buffer without co-solutes, negatively supercoiled DNA absorbs some of the tortional stress by forming locally melted DNA regions. The base-pairs in these locally melted regions are believed to adopt a configuration where nucleotide base pairing is disrupted. We find that the presence of dehydrating co-solutes like glycerol and ethylene glycol results in further destabilization of base-pairs in negatively supercoiled DNA. The presence of polyethylene glycol, commonly used as crowding agents, suppresses local strand separation and results in plectoneme formation even when DNA is negatively supercoiled. The results presented in this letter suggest many further directions for studies of DNA supercoiling and supercoiled DNA – protein interactions in molecular conditions that approximatein-vivomolecular composition.SIGNIFICANCEAccurate modelling of DNA mechanics is central to interpreting results of single molecule studies of DNA mechanics and DNA-protein interactions. While the effect of molecular conditions on short and relaxed DNA has been studied, the influence of molecular conditions on DNA supercoiling has not been explored. We present the first single molecule study of DNA supercoiling in presence of crowding and dehydrating co-solutes. We observe that co-solutes can increase or completely suppress stress-driven local strand separation in negatively supercoiled DNA. This change of DNA supercoiling is likely to significantly affect the function of DNA-binding proteins. Our results motivate the need for systematic exploration of DNA supercoiling in presence of co-solutes to accurately relatein-vitroDNA-protein interactions toin-vivoDNA-protein interactions.

show abstract

Effect of glycerol on free DNA: A molecular dynamics simulation study

Cited by 3 publications

References 26 publications

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

Deep-LASI: deep-learning assisted, single-molecule imaging analysis of multi-color DNA origami structures

Molecular Crowding Suppresses Mechanical Stress-Driven DNA Strand Separation

Contact Info

Product

Resources

About