Mechanisms of Small Molecule-DNA Interactions Probed by Single-molecule Force Spectroscopy

There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules

Strong DNA Deformation Required for Extremely Slow DNA Threading Intercalation by a Binuclear Ruthenium Complex

DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[μ‐bidppz‐(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes

Novel use of the Nintendo Wii board as a measure of reaction time: a study of reproducibility in older and younger adults

BACKGROUND: Reaction time (RT) has been associated with falls in older adults, but is not routinely tested in clinical practice. A simple, portable, inexpensive and reliable method for measuring RT is desirable for clinical settings. We therefore developed a custom software, which utilizes the portable and low-cost standard Nintendo Wii board (NWB) to record RT. The aims in the study were to (1) explore if the test could differentiate old and young adults, and (2) to study learning effects between test-sessions, and (3) to examine reproducibility. METHODS: A young (n = 25, age 20–35 years, mean BMI of 22.6) and an old (n = 25, age ≥65 years, mean BMI of 26.3) study-population were enrolled in this within- and between-day reproducibility study. A standard NWB was used along with the custom software to obtain RT from participants in milliseconds. A mixed effect model was initially used to explore systematic differences associated with age, and test-session. Reproducibility was then expressed by Intraclass Correlation Coefficients (ICC), Coefficient of Variance (CV), and Typical Error (TE). RESULTS: The RT tests was able to differentiate the old group from the young group in both the upper extremity test (p < 0.001; −170.7 ms (95%CI −209.4;-132.0)) and the lower extremity test (p < 0.001; −224.3 ms (95%CI −274.6;-173.9)). Moreover, the mixed effect model showed no significant learning effect between sessions with exception of the lower extremity test between session one and three for the young group (−35,5 ms; 4.6 %; p = 0.02). A good within- and between-day reproducibility (ICC: 0.76-0.87; CV: 8.5-12.9; TE: 45.7-95.1 ms) was achieved for both the upper and lower extremity test with the fastest of three trials in both groups. CONCLUSION: A low-cost and portable reaction test utilizing a standard Nintendo wii board showed good reproducibility, no or little systematic learning effects across test-sessions, and could differentiate between young and older adults in both upper and lower extremity tests

A General Mechanism for Competitor-induced Dissociation of Molecular Complexes

The kinetic stability of non-covalent macromolecular complexes controls many biological phenomena. Here we find that physical models of complex dissociation predict that competitor molecules will, in general, accelerate the breakdown of isolated bimolecular complexes by occluding rapid rebinding of the two binding partners. This prediction is largely independent of molecular details. We confirm the prediction with single-molecule fluorescence experiments on a well-characterized DNA strand dissociation reaction. Contrary to common assumptions, competitor-induced acceleration of dissociation can occur in biologically relevant competitor concentration ranges and does not necessarily imply ternary association of competitor with the bimolecular complex. Thus, occlusion of complex rebinding may play a significant role in a variety of biomolecular processes. The results also show that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatiotemporal resolution

FRNDY: A WOMEN SAFETY MOBILE APPLICATION

Women's personal security has always been a major concern, and numerous potential solutions have been discussed regarding how technology can be used to address the issue. Because of the rapid rise in smartphone usage, it is now possible to integrate personal security effectively using both hardware and software. Existing security methods necessitate some form of human input and the use of only pre-selected contacts. Many tragic occurrences have occurred in the case of women

A Critical Examination Of Planning Issues Surrounding The Formulation Of Public-Private Partnership Toll Road Projects In Malaysia

In Malaysia since the mid 1980s, Public-Private Partnership (PPP) toll road projects have been implemented as an alternative road system to the existing roads. Although it has been three decades since PPP toll road projects began to be implemented in Malaysia, the government could not implement certain terms in the contract resulting the government to compensate the private companies in the form of cash or in kinds (extension of concession period, tax exemptions, etc). This research was undertaken to investigate the changes within and among the social relations of actors in the public sector that promoted alterations in the formulation of PPP toll road projects in Malaysia. It aims to understand how changes are advocated which alter the dual relationship between social and institutional structures and individual agency in the production and reproduction of institutions. The institutional context for actors’ agency is formed by the policy, legal and regulatory and organisational frameworks. Over time in the PPP process, changes have taken place in these three frameworks which promoted and motivated changes in actors’ assumptions and rationales for action. These changes have resulted in alterations to the terms of the Concession Agreement (the agreement which stipulates the obligations between the public sector and private sector which governs the PPP toll road projects until the end of the concession period) which are being used for all the new PPP toll road project negotiations. Since this research is interested in the establishment and changes to actor’s social relations which are influenced by institutions, institutional theory is employed, while structuration theory provides a lens to look into changes over time

Diabetic Ketoacidosis Precipitated by COVID-19: A Case Report

Introduction: Diabetic emergencies such as diabetic ketoacidosis (DKA) are life-threatening complications, often precipitated by infections or illnesses such as COVID-19. Case presentation: A 55-year-old African American female presented to their primary care physician, complaining of fatigue, dehydration, decreased appetite, hypersomnia, and sudden weight loss, and a past medical history of Type 2 diabetes. They had a glucose level of >15 mmol/L and ketone level of >16 mmol/L; they were immediately sent to the emergency department for assessment of DKA. There, the patient tested positive for COVID-19. They had a glucose level of 361 mg/dL, a pH of 7.11, a bicarbonate level of 10 mEq/L, a sodium level of 125 mEq/L, a potassium level of 3.9 mEq/L, a chloride level of 95 mEq/L, an anion gap of 20, and a positive ketone level. Over the next few days, the patient’s condition got worse; their chest CT scan showed ground-glass opacities with consolidations in the middle and inferior lobes of the lungs bilaterally, along with interlobular septal thickening, which are consistent with an atypical infection, respiratory distress, and pneumonia. The patient was on intravenous fluids, insulin therapy and empirical antibiotics for the next few weeks, and eventually recovered. Discussion: Factors precipitating DKA in patients with diabetes in the setting of COVID-19are: the increased secretions of stress hormones that counter the effects of insulin and increase blood glucose levels, and the ways in which severe acute respiratory syndrome coronavirus 2 interacts with human cells, leading to pancreatic islet cell damage. Conclusion: Diabetes and COVID-19 intensify each other’s complications in patients diagnosed with both