Recently, numerous biological macromolecular experiments have been conducted with optical tweezers. For the single molecular stretching experiment with optical tweezers, three ways to determine the initial adhesion po...Recently, numerous biological macromolecular experiments have been conducted with optical tweezers. For the single molecular stretching experiment with optical tweezers, three ways to determine the initial adhesion point of DNA on the coverslip are described in this work. In addition, a new method through analyzing the displacement variance of the trapped particle to obtain the trap height is introduced. Using our proposed methods, the obtained force-extension curve for the operated dsDNA agrees well with the worm-like chain model. These improved methods are also applicable to other related biological macromolecular experiments requiring high precision.展开更多
Toroid formation is an important mechanism underlying DNA condensation, which has been investigated extensively by single-molecule experiments in vitro. Here, the de-condensation dynamics of DNA condensates were studi...Toroid formation is an important mechanism underlying DNA condensation, which has been investigated extensively by single-molecule experiments in vitro. Here, the de-condensation dynamics of DNA condensates were studied using magnetic tweezers combined with Brownian dynamics simulations. The experimental results revealed a surprising nonmonotonic dependence of the unfolding rate on the force applied under strong adhesion conditions, resembling the catchbond behavior reported in the field of ligand-receptor interactions. Simulation results showed that the different unfolding pathways of DNA condensate under large forces derive from the force-dependent deformation of the DNA toroid, which explains the catch-bond behavior of DNA condensate in the magnetic tweezers experiments. These results challenge the universality of the regular toroidal DNA unwrapping mechanism and provide the most complete description to date of multivalent cation-dependent DNA unwrapping under tension.展开更多
We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the len...We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the length of the DNA as a function of elapsed time. It is found that DNA condensates in a dynamic manner. The binding of hist, ones to DNA is energetically favoured, but the ten,sion applied on DNA tends to unravel the DNA-histone complex, The competition between the two processes determiners the rate of the DNA condensation.展开更多
The DNA gyrase of Escherichia coli plays an essential role in the life of this microorganism.It is unique among all topoisomerases because of its ability to introduce negative supercoils into DNA.This study investigat...The DNA gyrase of Escherichia coli plays an essential role in the life of this microorganism.It is unique among all topoisomerases because of its ability to introduce negative supercoils into DNA.This study investigated the single molecular interaction of E.coli gyrase with DNA using magnetic tweezers.The results showed that,in the absence of ATP,gyrase weakly binds the G and T segments.The stretched force of 0.7 pN can gradually destroy the binding,whereas that of 5.9 pN directly destroys it.Addition of high concentrations of norfloxacin enhances gyrase binding to both segments,making them adapt to 5.9 pN.DNA gyrase reduces the plectonemic dimension,which was determined by the bacterial enzyme and not by the pull force.Moreover,it has different affinities for positive supercoils,which it prefers,and negative supercoils.The time distribution of the dissociation of gyrase from DNA has a double-exponential form.We herein propose a model to explain this distribution and compare the results with those of other models.展开更多
Deoxyribonucleic acid(DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded(ss) strands, forming a double-stranded(ds) DNA with a right-handed double-helical confo...Deoxyribonucleic acid(DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded(ss) strands, forming a double-stranded(ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.展开更多
基金Supported by the National Natural Science Foundation of China under Grant No 91023049the National Basic Research Program of China under Grant No 2012CB937500
文摘Recently, numerous biological macromolecular experiments have been conducted with optical tweezers. For the single molecular stretching experiment with optical tweezers, three ways to determine the initial adhesion point of DNA on the coverslip are described in this work. In addition, a new method through analyzing the displacement variance of the trapped particle to obtain the trap height is introduced. Using our proposed methods, the obtained force-extension curve for the operated dsDNA agrees well with the worm-like chain model. These improved methods are also applicable to other related biological macromolecular experiments requiring high precision.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.1110434111474346+3 种基金11274374and 61275192)the National Key Basic Research Program of China(Grant No.2013CB837200)the Mechanobiology Institute at National University of Singapore
文摘Toroid formation is an important mechanism underlying DNA condensation, which has been investigated extensively by single-molecule experiments in vitro. Here, the de-condensation dynamics of DNA condensates were studied using magnetic tweezers combined with Brownian dynamics simulations. The experimental results revealed a surprising nonmonotonic dependence of the unfolding rate on the force applied under strong adhesion conditions, resembling the catchbond behavior reported in the field of ligand-receptor interactions. Simulation results showed that the different unfolding pathways of DNA condensate under large forces derive from the force-dependent deformation of the DNA toroid, which explains the catch-bond behavior of DNA condensate in the magnetic tweezers experiments. These results challenge the universality of the regular toroidal DNA unwrapping mechanism and provide the most complete description to date of multivalent cation-dependent DNA unwrapping under tension.
基金Supported by the National Natural Science Foundation of China under Grant No 10334100. The authors acknowledge the help of Professor .J. Yan of the National University of Singapore in setting up the instrument.
文摘We construct a system of magnetic tweezers and apply it to study the interaction between histones and DNA. The condensation of DNA by purified histones at low ionic strengths is directly monitored by recording the length of the DNA as a function of elapsed time. It is found that DNA condensates in a dynamic manner. The binding of hist, ones to DNA is energetically favoured, but the ten,sion applied on DNA tends to unravel the DNA-histone complex, The competition between the two processes determiners the rate of the DNA condensation.
基金supported by the National Natural Science Foundation of China (10834014,10974248)the National Basic Research Program of China (2009CB930704)
文摘The DNA gyrase of Escherichia coli plays an essential role in the life of this microorganism.It is unique among all topoisomerases because of its ability to introduce negative supercoils into DNA.This study investigated the single molecular interaction of E.coli gyrase with DNA using magnetic tweezers.The results showed that,in the absence of ATP,gyrase weakly binds the G and T segments.The stretched force of 0.7 pN can gradually destroy the binding,whereas that of 5.9 pN directly destroys it.Addition of high concentrations of norfloxacin enhances gyrase binding to both segments,making them adapt to 5.9 pN.DNA gyrase reduces the plectonemic dimension,which was determined by the bacterial enzyme and not by the pull force.Moreover,it has different affinities for positive supercoils,which it prefers,and negative supercoils.The time distribution of the dissociation of gyrase from DNA has a double-exponential form.We herein propose a model to explain this distribution and compare the results with those of other models.
基金supported by the Fundamental Research Funds for the Central Universities(Grant No.2013121005)the National Natural Science Foundation of China(Grant Nos.11474237 and 11574310)+1 种基金the 111 Project (Grant No.B16029)the National Research Foundation of Singapore through the NRF Investigatorship and the Mechanobiology Institute
文摘Deoxyribonucleic acid(DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded(ss) strands, forming a double-stranded(ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.
