Force-induced melting of DNA hairpin: Unfolding pathways and phase diagrams.

Using the exact enumeration technique, we have studied the force-induced melting of a DNA hairpin on the face centered cubic lattice for two different sequences which differ in terms of loop closing base pairs. The melting profiles obtained from the exact enumeration technique is consistent with the Gaussian network model and Langevin dynamics simulations. Probability distribution analysis based on the exact density of states revealed the microscopic details of the opening of the hairpin. We showed the existence of intermediate states near the melting temperature. We further showed that different ensembles used to model single-molecule force spectroscopy setups may give different force-temperature diagrams. We delineate the possible reasons for the observed discrepancies.

Compliance Assessment of Tobacco Products to Cigarettes and Other Tobacco Products Act in India with Reference to Third Amendment Rules, 2020.

OBJECTIVE: Tobacco use is associated with mortality in low- and middle-income countries including India with dual burden of smoking and smokeless tobacco (SLT). Aligning with the FCTC, India has made substantial amendments in strengthening graphic warning under Cigarettes and Other Tobacco Products Act (COTPA) for sections 7,8 9 and "Specified warning". Compliance assessment studies are necessary to understand current status of implementation for packaging laws. This study aimed to assess the compliance of COTPA sections 7,8 9 and Cigarettes and other Tobacco Products (Packaging and Labelling) Third Amendment Rules, 2020 in Delhi. METHODS: Cross-sectional study was conducted in two districts of Delhi selected by simple random sampling. Fifteen points of sales were selected from each district through purposive sampling and 57 smoking and smokeless tobacco products were collected with Indian and foreign origin. An observation checklist for product analysis was prepared and pack analysis done based on COTPA sections 7,8 and 9 along with Third Amendment,2020 which included pictures and warnings to be circulated in 2021. RESULT: Total 57 samples has smoking (49.1%), smokeless (50.9%) with no SLT product of foreign origin. SLT and foreign products had low compliance of Section 7 and third amendment 2020 rules which includes manufacturing date and origin. Indian smoking products were highly compliant to section 8 and 9 whereas foreign and SLT products showed low compliance to section 8. COTPA Third Amendment Rules (2020) compliance was seen in Indian products with regards to SW (68.4%), PW (61.4%) and quit line (78.9%) with no compliance at all for foreign products. CONCLUSION: Foreign brands and SLT products had low compliance with sections 7 and 8 of COTPA and its amendments (2020). Compliance with illicit trade and SW needs regulation and strict implementation of law for SLT products.

Can one detect intermediate denaturation states of DNA sequences by following the equilibrium open-close dynamic fluctuations of a single base pair?

Melting of DNA sequences may occur through a few major intermediate states, whose influence on the melting curve has been discussed previously, while their effect on the kinetics has not been explored thoroughly. Here, we chose a simple DNA sequence, forming a hairpin in its native (zipped) state, and study it using molecular dynamic (MD) simulations and a model integrating the Gaussian network model with bond-binding energies-the Gaussian binding energy (GBE) model. We find two major partial denaturation states, a bubble state and a partial unzipping state. We demonstrate the influence of these two states on the closing-opening base pair dynamics, as probed by a tagged bond auto-correlation function (ACF). We argue that the latter is measured by fluorescence correlation spectroscopy experiments, in which one base of the pair is linked to a fluorescent dye, while the complementary base is linked to a quencher, similar to the experiment reported by Altan-Bonnet et al. [Phys. Rev. Lett. 90, 138101 (2003)]. We find that tagging certain base pairs at temperatures around the melting temperature results in a multi-step relaxation of the ACF, while tagging other base pairs leads to an effectively single-step relaxation, albeit non-exponential. Only the latter type of relaxation has been observed experimentally, and we suggest which of the other base pairs should be tagged in order to observe multi-step relaxation. We demonstrate that this behavior can be observed with other sequences and argue that the GBE can reliably predict these dynamics for very long sequences, where MD simulations might be limited.

Manipulation of double-stranded DNA melting by force.

By integrating elasticity-as described by the Gaussian network model-with bond binding energies that distinguish between different base-pair identities and stacking configurations, we study the force induced melting of a double-stranded DNA (dsDNA). Our approach is a generalization of our previous study of thermal dsDNA denaturation [J. Chem. Phys. 145, 144101 (2016)JCPSA60021-960610.1063/1.4964285] to that induced by force at finite temperatures. It allows us to obtain semimicroscopic information about the opening of the chain, such as whether the dsDNA opens from one of the ends or from the interior, forming an internal bubble. We study different types of force manipulation: (i) "end unzipping," with force acting at a single end base pair perpendicular to the helix, (ii) "midunzipping," with force acting at a middle base pair perpendicular to the helix, and (iii) "end shearing," where the force acts at opposite ends along the helix. By monitoring the free-energy landscape and probability distribution of intermediate denaturation states, we show that different dominant intermediate states are stabilized depending on the type of force manipulation used. In particular, the bubble state of the sequence L60B36, which we have previously found to be a stable state during thermal denaturation, is absent for end unzipping and end shearing, whereas very similar bubbles are stabilized by midunzipping, or when the force location is near the middle of the chain. Ours results offer a simple tool for stabilizing bubbles and loops using force manipulations at different temperatures, and may implicate on the mechanism in which DNA enzymes or motors open regions of the chain.

Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies.

We study DNA denaturation by integrating elasticity - as described by the Gaussian network model - with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

Force induced melting of the constrained DNA.

We develop a simple model to study the effects of the applied force on the melting of a double stranded DNA (dsDNA). Using this model, we could study the stretching, unzipping, rupture and slippagelike transition in a dsDNA. We show that in absence of an applied force, the melting temperature and the melting profile of dsDNA strongly depend on the constrained imposed on the ends of dsDNA. The nature of the phase boundary of the force-temperature diagram, which separates the zipped and the open state for the shearinglike transition is remarkably different than the DNA unzipping.

Force induced unfolding of biopolymers in a cellular environment: a model study.

Effect of molecular crowding and confinement experienced by protein in the cell during unfolding has been studied by modeling a linear polymer chain on a percolation cluster. It is known that internal structure of the cell changes in time, however, they do not change significantly from their initial structure. In order to model this we introduce the correlation among the different disorder realizations. It was shown that the force-extension behavior for correlated disorder in both constant force ensemble and constant distance ensemble is significantly different than the one obtained in absence of molecular crowding.

Effects of molecular crowding on stretching of polymers in poor solvent.

We consider a linear polymer chain in a disordered environment modeled by percolation clusters on a square lattice. The disordered environment is meant to roughly represent molecular crowding as seen in cells. The model may be viewed as the simplest representation of biopolymers in a cell. We show the existence of intermediate states during stretching arising as a consequence of molecular crowding. In the constant distance ensemble the force-extension curves exhibit oscillations. We observe the emergence of two or more peaks in the probability distribution curves signaling the coexistence of different states and indicating that the transition is discontinuous unlike what is observed in the absence of molecular crowding.