凝血酶适体DNA四螺旋构象去折叠机制的NMR研究

凝血酶适体DNA\[thrombin binding DNA aptamer d (G₁G₂T₃T₄G₅G₆T₇G₈T₉G₁₀G₁₁T₁₂T₁₃G₁₄G₁₅),TBA]是对凝血酶有极高亲和性、并能有效抑制凝血酶凝血功能的单链DNA适体. 凝血酶适体DNA在K⁺等离子存在时呈现出椅式构象,其中2个堆积的四碱基体（G-quartet）构成椅子的主体部分,而1个TGT loop环和2个TT loop环分别构成椅子的靠背和椅脚. 我们测定了在K⁺存在时凝血酶适体DNA中亚氨基质子的交换速率, 发现位于TGT loop环和TT loop环内的亚氨基质子G6、G5和G14由于受TGT loop环和TT loop环的保护有比较小的交换速率,而位于环之外的亚氨基质子G2、G11和G15有较大的交换速率;TGT loop环稳定性同TT loop环相似;碱基T4、T13和T9在稳定凝血酶适体DNA结构中起着很大的作用. 这些证据进一步支持了凝血酶适体DNA的去折叠机制：位于TGT loop环和TT loop环之外的碱基对G1G15、G2G14和G5G11首先断裂其Hoogsteen氢键,而TGT loop环、TT loop环和其内的Hoogsteen氢键保持完好;当温度进一步升高时,TGT loop环和两个TT loop环打开环状结构,凝血酶适体DNA的椅式构象彻底解体,转变为自由单链.      

Methylene blue as a DNA probe for a comparative study of Cd, Pb and Cr ions binding to calf thymus DNA

Methylene blue (MB) was developed as a sensitive DNA probe for a comparative study of Cd²⁺, Pb²⁺ and Cr³⁺ ions binding with calf thymus DNA (ctDNA). The fluorescence intensity of the MB-ctDNA system increased dramatically when heavy metal ions (Cd²⁺, Pb²⁺ and Cr³⁺ ions) were added, which indicated that some of the bound MB molecules were released from the ctDNA base pairs. To compare the binding affinity of these three different heavy metal ions with ctDNA, the relationships between the fluorescence intensity of the MB-ctDNA-M (Metal ions) system and the concentration ratio of [M]/[DNA(p)] were investigated. The results showed that the order of the binding affinity of heavy metal ions with ctDNA had the following sequence: Cr³⁺> Cd²⁺>Pb²⁺. This order was further proved by the effects of heavy metal ions on the number of MB bound to ctDNA, the measurements of binding constants of these heavy metal ions to ctDNA, and the effects of heavy metal ions on the absorption of the MB-ctDNA system. In addition, the interaction mechanisms of Cd²⁺, Pb²⁺ and Cr³⁺ ions with ctDNA were also discussed in detail. These results indicated that their interaction mechanisms are related to the concentration ratios of heavy metal ions to DNA.     

Influence of Mg+2 and Cu+2 on the Interaction Between Quinolone and Calf Thymus DNA

Mg⁺² and Cu⁺² have different binding capacities to quinolone drugs and have different binding modes with calf thymus DNA. Using the method of absorption and fluorescence spectroscopy, the influence of Mg⁺² and Cu⁺² on the binding between calf thymus DNA and each of four quinolone drugs has been studied. The results show that both Mg⁺² and Cu⁺² can bind with the four drugs. In the absence of divalent metal ions, quinolone drugs interact with DNA double helix by forming hydrogen bonds between the carboxyl and carbonyl groups of the drugs and the phosphate groups of the DNA bases, and the binding capacity shows a close relationship with the drug structures. The two metal ions show different influences on the binding between the drug and DNA, which depends on the type of ion, concentration of the metal ions and the structure of drugs. Mg⁺² in lower concentrations (0.01 mM to 3.0 mM) can act as a bridge between the carboxyl group/carbonyl group of the drug and the phosphate group of the DNA by electrostatic interaction, while Cu⁺² can act as an intermediary ion between carboxyl group/carbonyl group of the drug and the DNA bases by a co-ordinate bond. Both actions can increase the interaction of the ?? electron between the condensed rings of the drugs and the DNA bases. In some conditions, Cu⁺² can weaken the binding between the drug and the DNA by competitive inhibition if there is a site on the drug that can directly bind both DNA bases and Cu⁺².     

Molecular dynamics simulations of A-DNA in bivalent metal ions salt solution

A-form DNA is one of the biologically active double helical structure. The study of A-DNA structure has an extensive application for developing the field of DNA packaging in biotechnology. In aqueous solution, the A-DNA structure will have a free transformation, the A-DNA structure will be translated into B-form structure with the evolution of time, and eventually stabilized in the B-DNA structure. To explore the stability function of the bivalent metal ions on the A-DNA structure, a series of molecular dynamics simulations have been performed on the A-DNA of sequence (CCCGGCCGGG). The results show that bivalent metal ions (Mg²⁺, Zn²⁺, Ca²⁺) generate a great effect on the structural stability of A-DNA in the environment of high concentration. As the interaction between metal ions and electronegative DNA chains, the stability of A-DNA in solution is gradually improved with the increasing solution concentration of ions. In metal salt solution with high concentration, metal ions can be easily distributed in the solvation shells around the phosphate groups and further lead to the formation of shorter and more compact DNA structure. Also, under the condition of the same concentration and valency of the metal ions, the stability of A-DNA structure is different. The calculations indicate that the structure of A-DNA in CaCl₂ solution is less stable than in MgCl₂ and ZnCl₂ solution.     

The elastic theory of a single DNA molecule

We study the elastic responses of double-(ds) and single-stranded (ss) DNA at external force fields. A double-strand-polymer elastic model is constructed and solved by path integral methods and Monte Carlo simulations to understand the entropic elasticity, cooperative extensibility, and supercoiling property of dsDNA. The good agreement with experiments indicates that short-ranged base-pair stacking interaction is crucial for the stability and the high deformability of dsDNA. Hairpin-coil transition in ssDNA is studied with generating function method. A threshold force is needed to pull the ssDNA hairpin patterns, stabilized by base pairing and base-pair stacking, into random coils. This phase transition is predicted to be of first order for stacking potential higher than some critical level, in accordance with experimental observations.     

Interaction between human telomeric G-quadruplexes characterized by single molecule magnetic tweezers

Human telomeric G-quadruplex plays a crucial role in regulating the genome stability. Despite extensive studies on structures and kinetics of monomeric G-quadruplex, the interaction between G-quadruplexes is still in debate. In this work,we employ magnetic tweezers to investigate the folding and unfolding kinetics of two contiguous G-quadruplexes in 100-mM K~+buffer. The interaction between G-quadruplexes and the consequent effect on the kinetics of G-quadruplex are revealed. The linker sequence between G-quadruplexes is further found to play an important role in the interaction between two G-quadruplexes. Our results provide a high-resolution insight into kinetics of multimeric G-quadruplexes and genome stability.     

光谱及等温滴定量热法探究盐酸阿霉素与DNA作用机理

利用荧光光谱法,紫外可见光谱法,红外光谱法,圆二色谱法和等温滴定量热法等手段,对抗癌药物盐酸阿霉素 (DOX) 与DNA的作用过程进行研究,测得了它们的结合常数K_a、结合位点数n、反应焓变ΔH、熵变ΔS及ΔG,且在结合过程中,B型DNA的螺旋结构在一定程度发生改变。荧光光谱的数据显示出显著的猝灭效应, 表明DNA是一个DOX荧光的很好的猝灭剂。红外光谱表明阳离子DOX+通过静电吸引与 DNA 的磷酸基团相互作用,且DOX的碳氢链通过疏水缔合与DNA作用。ITC测定了DOX和DNA相互作用的焓变和熵变,表明DOX的烃链和DNA的碱基之间的疏水性相互作用提供了结合的驱动力。     

Ultraviolet Spectroscopic Study of Cu2+ Induced DNA Melting at Room Temperature

Complexes of Calf thymus DNA with Cu²⁺ ions have been studied using ultraviolet (UV) absorption spectroscopy to probe the binding and melting behavior of DNA in the complex at 25°C, causing a melting of DNA as effectively as heating to 100°C. This strengthens the concept of Cu²⁺ binding to the DNA bases as recently demonstrated by Richard et al.¹     

Metal ion interaction with ribosomal RNA

Summary We have used UV differential spectroscopy in order to detect small modifications in the ribosomal RNA absorption spectrum due to the binding of rRNA molecules with the metal ions Na⁺, K⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺. Our data show that all the ions, investigated are involved in ion-type bond with the phosphate groups of rRNA and cause a refolding of the molecules with an overall increase in basebase ?stacking? interactions. Besides this ion-type binding with phosphate groups, transition metal ions Mn²⁺, Co²⁺, and Ni²⁺ are also able to bond directly to the bases To speed up publication, the authors of this paper have agreed to not receive the proofs for correction.     

Computational determination of radiation damage effects on DNA structure

Molecular dynamics (MD) studies of several radiation originated lesions on the DNA molecules are presented. The pyrimidine lesions (cytosinyl radical, thymine dimer, thymine glycol) and purine lesion (8-oxoguanine) were subjected to the MD simulations for several hundred picoseconds using MD simulation code AMBER 5.0 (4.0). The simulations were performed for fully dissolved solute molecules in water. Significant structural changes in the DNA double helical structure were observed in all cases which may be categorized as: a) the breaking of hydrogen bonds network between complementary bases and resulted opening of the double helix (cytosinyl, radical, 8-oxoguanine); b) the sharp bending of the DNA helix centered at the lesion site (thymine dimer, thymine glycol); and c) the flippingout of adenine on the strand complementary to the lesion (8-oxoguanine). These changes related to the overall collapsing of the double helical structure around the lesion, are expected to facilitate the docking of the repair enzyme into the DNA in the formation of DNA-enzyme complex. The stable DNA-enzyme complex is a necessary condition for the onset of the enzymatic repair process. In addition to structural changes, specific values of electrostatic interaction energy were determined at several lesion sites (thymine dimer, thymine glycol and 8-oxoguanine). This lesion-specific electrostatic energy is a factor that enables repair enzyme to discriminate lesion from the native site during the scanning of the DNA surface.     

Influence of amphotericin B on liquid crystal state of the Cholesterol/Dipalmitoylphosphatidylcholine monolayer in the presence of different metal cations

Amphotericin B is a very effective antifungal drug,but it has an adverse reaction to the membrane of mammals' cells.The interaction between Am B and cholesterol(Chol) causes the formation of pores on the membrane to destroy its integrity.In particular,Am B has a significant effect on the permeability of membrane for K~+ions.It has been reported that Na+ions and Ca~(2+)ions may have some influence on the interaction between amphotericin B and lipid molecules.In this work,the effects of these metal cations on the physical state and intermolecular interaction of the Cholesterol/Dipalmitoylphosphatidylcholine(Chol/DPPC) monolayer with and without Am B have been investigated.The addition of Am B induces the change of physical state of the lipid monolayer from liquid-gel phase to liquid phase.Different metal cations could influence the phase transition of the Am B-lipid monolayer.The K~+ions and Ca2+ions make the obvious phase transition disappear.However,the presence of Na+ions has little influence on the phase transition of the Am B-lipid monolayer.The addition of Am B and the presence of different metal cations weaken the attractive force on the monolayers.After addition of Am B,the force between the molecules is the strongest in the environment of K+ions,thus is the weakest in the environment of Ca~(2+)ions,which may be due to the distribution of these metal cations inside and outside of cells.A large number of K+ions distribute inside of the cells,thus most of Na+and Ca~(2+)ions exist out of the cells.Hence,it may be possible that when Am B molecules are out of the cells,the reaction between the drug and lipid molecules is weaker than that inside the cells.These results may have a great reference value for further studying the toxicity mechanism of Am B and the influence of metal cations on the membrane.     

Raman spectroscopic studies on the conformational changes of calf thymus DNA induced by Cd2+ ions

We used Raman spectroscopy to study the conformational changes of DNA induced by Cd2+ ions in different Cd2+ concentrations solution. The experimental results show that when the Cd2+/PO-2 ratio R increased from 0 to 3.0, the band 835.0 cm-1 shifted about 8 cm-1, and the overlapping spectra of 1446.0and 1461.0 cm-1 separated and moved to 1441.0 and 1458.0 cm-1, respectively. This indicates that the conformation of DNA has changed from a "normal" B-form to a "modified" B'-form. At the same time,changes of other bands demonstrate that parts of base stacking collapse and some hydrogen bonds between AT are disrupted, AT base pairs are damaged more larger than GC base pairs.     

Self-organised criticality in base-pair breathing in DNA with a defect

We analyse base-pair breathing in a DNA sequence of 12 base-pairs with a defective base at its centre. We use both all-atom molecular dynamics (MD) simulations and a system of stochastic differential equations (SDEs). In both cases, Fourier analysis of the trajectories reveals self-organised critical behaviour in the breathing of base-pairs. The Fourier Transforms (FTs) of the inter-base distances show power-law behaviour with gradients close to −1. The scale-invariant behaviour we have found provides evidence for the view that base-pair breathing corresponds to the nucleation stage of large-scale DNA opening (or ‘melting’) and that this process is a (second-order) phase transition. Although the random forces in our SDE system were introduced as white noise, FTs of the displacements exhibit pink noise, as do the displacements in the AMBER/MD simulations.     

金属离子诱导下DNA折叠的不同机理

利用等温滴定量热仪、圆二色谱和荧光光谱,研究和分析了人工合成的DNA单链序列d(T₆C₆T₆C₆T₆C₆T₆)与Hg²⁺和Ag⁺相互作用的折叠过程. 在改变离子添加顺序的情况下,尽管热力学数据显示两种情况下都能通过两种不同反应路径得到一种相对稳定的发夹结构,但等温滴定量热仪数据却显示最终产物形成的机理截然不同.当先加入Hg²⁺时,发夹结构首先通过T-Hg-T碱基对形成然后C-Ag-C碱基对得到进一步稳定. 然而当先加入Ag⁺时,通过圆二色谱和荧光分析确认了一种不常见的金属碱基对T-Ag-C取代了经典的C-Ag-C碱基对.     

Effects of metal ion binding on an oncomodulin mutant containing a novel calcium-binding loop

The Ca²⁺-binding protein oncomodulin was altered by cassette mutagenesis of the CD site (CDOM33) with a sequence that was derived by a consensus method using over 250 known Ca²⁺-binding loop sequences. This mutant was studied using time-resolved and steady-state fluorescence from the Trp residue included at position 7 of the loop (position 57 of the protein sequence). The fluorescence characteristics of this species in the absence and presence of metal ions were compared to those of a tetradecapeptide containing the loop and the single Trp mutant of oncomodulin, Y57W. The fluorescence properties of CDOM33 were quite different from the peptide, both in the apo form and in response to metal binding. The consensus CD loop in CDOM33 exhibited the characteristics of a Ca²⁺/Mg²⁺ site in contrast to the Ca²⁺ specificity of the wild-type CD loop. The Trp analogue, 5-hydroxytryptophan (5HW), was incorporated into both oncomodulin mutants to produce Y75(5HW) and 5HW-CDOM33. Results showed that this intrinsic probe was relatively insensitive to structural changes in the mutants upon metal binding compared to Trp itself.     

A first-principles study on DNA sequencing using graphene quantum dot

In this paper, we propose a new device based on graphene quantum dot (GQD) to interrogate nucleotide in a DNA molecule. We have conducted non-equilibrium Green’s function together with the density functional theory simulations to show zero transmission curves for a system which includes nucleobases. The simulation results indicates several characteristic peaks in the electron transmission curve for any single base on the quantum dot which can be utilized to distinguish between bases. Number and positions of the peaks, as well as their amplitude, depend on the type of the bases and their relative position to the dot. Thus, this structure shows remarkable distinction characteristic in zero bias transmission curve and can yield minimal ambiguity in the adenine, cytosine, guanine and thymine nucleotide detection. Due to stacking of nucleobases on the graphene and consequent attenuation in directional fluctuations, stable measurement is also expected.     

Interaction mechanism of 2-aminobenzothiazole with herring sperm DNA

The toxic interaction of 2-aminobenzothiazole (2-ABT) with herring sperm DNA (hs-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated that 2-ABT interacted with hs-DNA in a minor groove binding mode. The binding constant and the number of binding sites were 7.2×10³ L mol^?1 and 0.95, respectively. Circular dichroism spectroscopy (CD) was employed to measure the conformation change of hs-DNA in the presence of 2-ABT, which verified the minor groove binding mode. The molecular modeling results illustrated that 2-ABT tended to bind in the region of rich A–T base pairs through the hydrogen bond between A 18 and amino group of 2-ABT. Sequence specificity was confirmed by comparison on the interactions of 2-ABT with four kinds of bases. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with biomacromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.     

血卟啉单甲醚对DNA损伤的拉曼光谱研究

利用激光拉曼光谱技术研究了新型光敏剂血卟啉单甲醚对ＤＮＡ（脱氧核糖核酸）分子的光动力学损伤。结果表明：ＤＮＡ分子由典型Ｂ型构型变成了一种修饰过的Ｂ型。连接碱基对的氢键被部分打断,碱基堆叠也有所与解。碱基本身也遭到了破坏,有脱氨基现象发生。腺嘌呤和胞嘧啶对血咔啉单甲醚的光动力学损伤更敏感。     

Stationary solutions for a modified Peyrard-Bishop DNA model with up to third-neighbor interactions

We investigate a DNA model that takes into account stacking interactions with neighbors up to three bases away. The model is a generalization of the well-known Peyrard-Bishop (PB) model and is motivated by studies that suggest that nearest-neighbor models for base-pair interaction in a DNA chain might not be enough to capture the mechanism and dynamics of DNA base-pair opening. We study stationary solutions of the modified model and investigate their stability. A comparison with the PB model reveals that under a wide range of parameter values the main characteristics of the original model --such as the hyperbolicity of the equilibrium at the origin-- are preserved, but new types of stationary solutions emerge.