Cooperative Melting Transition Research Articles

Base-specific pre-melting and melting transitions of DNA in presence of ionic liquids probed by synchrotron-based UV resonance Raman scattering

Hydrated ionic liquids (ILs) have been identified as solvent media able to enhance the structural stability of deoxyribonucleic acid (DNA). In this work, we investigate the molecular interaction between imidazolium-based ILs and DNA during its thermal unfolding pathway, by exploiting synchrotron-based UV Resonance Raman scattering (UVRR) experiments. This technique gives a selective focus on the thermal responses of specific nucleobases in the structure of DNA, providing the experimental sensitivity to both cooperative and local structural changes occurring along the complex unfolding process of DNA. UVRR measurements probe two distinct temperature-dependent phenomena occurring in the DNA double-helix, i.e. a non-cooperative pre-melting mainly involving adenine bases and a cooperative melting transition primarily localized on guanine tracts. The analysis of Raman spectra reveals that both the cation and anion of the ionic liquids strongly interact with the structure of DNA, thus affecting the melting process but not perturbing the pre-melting transition that precedes the complete separation of the strands of DNA. Overall these results suggest that the dominant interaction occurs between the imidazolium cation and the bases of guanine and thymine in the structure of DNA, in agreement with previous results of molecular dynamics simulations.

Spectroscopic study on the binding of chelerythrine with duplex poly (rA): A model of RNA intercalation

Here we have reported a detail study on the interaction of the benzophenanthridine alkaloid chelerythrine (CHL) with double stranded polyriboadenylic acid [ds poly (rA)] by exploiting various spectroscopic techniques. The alkaloid shows high binding affinity (binding constant is 1.10×105M−1) towards the double stranded RNA as revealed from Scatchard plot. The binding was confirmed by hypochromic effect in the UV–vis spectrum of CHL, increase in fluorescence intensity of CHL and perturbations of the circular dichroism (CD) spectrum of ds poly (rA). Later fluorescence quenching, cooperative CD melting transition, viscometric and molecular modeling studies establish the fact that the alkaloid binds to the ds poly (rA) by the mechanism of intercalation. Thermodynamic parameters obtained from the isothermal titration calorimetric (ITC) study show that the binding is favoured by negative enthalpy and small positive entropy changes. This report may be a model for intercalation of small molecule like CHL to the double stranded RNA.

Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid conjugates for antisense gene regulation.

Herein, we report the synthesis of DNA-functionalized infinite-coordination-polymer (ICP) nanoparticles as biocompatible gene-regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3-hydroxy-4-pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe(III) to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe(III)-HOPO ICP particles by copper-free click chemistry afforded colloidally stable nucleic-acid nanoconstructs. The DNA-ICP particles, when cross-linked through sequence-specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA-ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.

Biocompatible Infinite‐Coordination‐Polymer Nanoparticle–Nucleic‐Acid Conjugates for Antisense Gene Regulation

AbstractHerein, we report the synthesis of DNA‐functionalized infinite‐coordination‐polymer (ICP) nanoparticles as biocompatible gene‐regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3‐hydroxy‐4‐pyridinone (HOPO) ligand bearing a pendant azide. Addition of FeIII to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the FeIII–HOPO ICP particles by copper‐free click chemistry afforded colloidally stable nucleic‐acid nanoconstructs. The DNA–ICP particles, when cross‐linked through sequence‐specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA–ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.

Internal deletion compromises the stability of dystrophin

Duchenne muscular dystrophy (DMD) is a deadly and common childhood disease caused by mutations that disrupt dystrophin protein expression. Several miniaturized dystrophin/utrophin constructs are utilized for gene therapy, and while these constructs have shown promise in mouse models, the functional integrity of these proteins is not well described. Here, we compare the biophysical properties of full-length dystrophin and utrophin with therapeutically relevant miniaturized constructs using an insect cell expression system. Full-length utrophin, like dystrophin, displayed a highly cooperative melting transition well above 37°C. Utrophin constructs involving N-terminal, C-terminal or internal deletions were remarkably stable, showing cooperative melting transitions identical to full-length utrophin. In contrast, large dystrophin deletions from either the N- or C-terminus exhibited variable stability, as evidenced by melting transitions that differed by 20°C. Most importantly, deletions in the large central rod domain of dystrophin resulted in a loss of cooperative unfolding with increased propensity for aggregation. Our results suggest that the functionality of dystrophin therapeutics based on mini- or micro-constructs may be compromised by the presence of non-native protein junctions that result in protein misfolding, instability and aggregation.

Single Molecule DNA Interactions of the Nucleic Acid Chaperone Protein from the Line-1 Retrotransposon

Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and nucleic acid aggregation, duplex destabilization, and rapid protein binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as ORF1p from the retrotransposon LINE-1, are not as well understood. We used single molecule DNA stretching in combination with site-directed mutagenesis of ORF1p as a method for detailed characterization of its chaperone activity. Wild type ORF1p significantly reduces the cooperativity of the force-induced melting transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA), indicating that DNA melting is more easily initiated in its presence. ORF1p also aggregates both dsDNA and ssDNA, and exhibits relatively rapid binding kinetics. Altering certain residues has dramatic effects on chaperone activity. Stretching curves in the presence of mutant R284A, which is inactive in retrotransposition assays, exhibit a cooperative melting transition and minimal DNA aggregation. Retrotransposition is partially restored with mutant R284K, which alters the melting transition and strongly aggregates ssDNA. Similarly, mutant Y318A has minimal retrotransposition activity, and stretching curves reflect only a small change in melting transition cooperativity. The Y318F mutant, which largely restores retrotransposition, alters the melting transition in a way similar to wild type ORF1p. Thus, DNA stretching results indicate that reduced cooperativity of the melting transition is associated with greater nucleic acid chaperone and retrotransposition activity of ORF1p variants.

PLA2 Type IIA Increases Platelet Plasma Membrane Rigidity During Cold Induced Activation

There is a wide discussion regarding the origin and function of lipid domains in human cells. An example in which lipid domains may play a functional role is the plasma membrane of human platelets. Platelets are susceptible to chilling, activating when the temperature falls below 20 °C. This limitation represents a crucial issue in terms of storage of platelets. It has been previously shown that cold-induced platelet activation is correlated with the formation of macroscopic lipid domains during cooling. This is a result of the fact that platelets present low cholesterol content (15 mol%), which results in the presence of a cooperative lipid melting transition centered at 15°C. This transition is responsible for the formation of the macroscopic lipid domains during cooling. Human secretory phopholipase A2 type IIA (sPLA2-IIA) catalyses the hydrolysis of the sn-2 ester bond in glycerolipids to produce fatty acids and lysolipids. Recently we have shown that its activity is triggered by the local enrichment of anionic lipids in fluid domains during phase coexistence. Since human platelets expose their anionic lipid content during chilling, we propose that PLA2-IIA is triggered during this process, and that the activity of the enzyme on the fluid domains induces an overall increase in rigidity in the platelet plasma membrane due to an increase in the proportion of ordered lipids. We present a combined FTIR and Laurdan fluorescence spectroscopy study on the thermotropic phase behavior of human platelets, and show that during cold-induced activation membrane rigidity increases. We present results on how the activity of sPLA2-IIA is regulated by the thermotropic phase behavior of the platelet plasma membrane after platelets have been artificially activated through sonication. We show that PLA2-IIA activity increases the rigidity of these activated platelet plasma membranes.

Synthetically programmable DNA binding domains in aggregates of DNA-functionalized gold nanoparticles.

Polyvalent DNA-functionalized gold nanoparticle conjugates (DNA–Au NPs) have proven useful in a variety of assembly,[1–3] biodiagnostic,[4–6] and nanotherapeutic[7–10] applications. Their widespread use is a consequence of: 1) their novel hybridization properties and 2) straightforward methods for synthesizing macroscopic quantities of them in relatively monodisperse form.[1,11,12] In some applications, the utility of DNA–Au NPs relies on their ability to assemble via DNA hybridization into polymeric aggregates (Scheme 1A).[1] This reaction is accompanied by a concomitant red-to-blue color change, a consequence of the dampening and red-shifting of the nanoparticle surface plasmon resonance (SPR) band at ~520 nm (for a 15-nm nanoparticle).[13] As the temperature is increased above the melting temperature of the duplex DNA linkages connecting the gold nanoparticles, the polymeric structure dehybridizes, the spectroscopic signature associated with the dispersed particles is restored, and a single, highly cooperative melting transition is observed. The melting transition occurs at a higher temperature and over a more narrow temperature range than free duplex DNA of the same sequence.[1,14,15]

Nucleic Acid Therapeutic Carriers with On-Demand Triggered Release

Biohybrid platforms such as synthetic polymer networks engineered from artificial and natural materials hold immense potential as drug and gene delivery vehicles. Here, we report the synthesis and characterization of novel polymer networks that release oligonucleotide sequences via enzymatic and physical triggers. Chemical monomers and acrylated oligonucleotides were copolymerized into networks, and phosphoimaging revealed that 70% of the oligonucleotides were incorporated into the networks. We observed that the immobilized oligonucleotides were readily cleaved when the networks were incubated with the type II restriction enzyme BamHI. The diffusion of the cleaved fragments through the macromolecular chains resulted in relatively constant release profiles very close to zero-order. To our knowledge, this is the first study which harnesses the sequence-specificity of restriction endonucleases as triggering agents for the cleavage and release of oligonucleotide sequences from a synthetic polymer network. The polymer networks exhibited an oligonucleotide diffusion coefficient of 5.6 x 10(-8) cm(2)/s and a diffusional exponent of 0.92. Sigmoidal temperature responsive characteristics of the networks matched the theoretical melting temperature of the oligonucleotides and indicated a cooperative melting transition of the oligonucleotides. The networks were also triggered to release a RNA-cleaving deoxyribozyme, which degraded a HIV-1 mRNA transcript in vitro. To tailor release profiles of the oligonucleotides, we controlled the structure of the macromolecular architecture of the networks by varying their cross-linking content. When incubated with DNase I, networks of cross-linking content 0.15%, 0.22%, and 0.45% exhibited oligonucleotide diffusion coefficients of 1.67 x 10(-8), 7.65 x 10(-9), and 2.7 x 10(-9) cm(2)/s, and diffusional exponents of 0.55, 0.8, and 0.8, respectively. The modular nature of our platform promises to open new avenues for the creation and optimization of a rich toolbox of novel drug and gene delivery platforms. We anticipate further inquiry into nucleic acid based programmable on-demand switches and modulatory devices of exquisite sensitivity and control.

Specific binding and self-structure induction to poly(A) by the cytotoxic plant alkaloid sanguinarine

The cytotoxic plant alkaloid sanguinarine was found to bind preferentially and strongly to single stranded poly(A) with an association constant (K a) in the range 3.6–4.6 × 10 6 M − 1 in comparison to several nucleic acids. The binding induced unique self-structure formation in poly(A) that showed cooperative melting transition in circular dichroism, absorbance, and differential scanning calorimetry studies. The alkaloid binding was characterized to be intercalation as revealed from fluorescence quenching experiments and was predominantly enthalpy driven as revealed from isothermal titration calorimetry. Sanguinarine is the first and only natural product so far known to induce a self-structure formation in poly(A).

Toward beta-amino acid proteins: a cooperatively folded beta-peptide quaternary structure.

Folded polymers in nature are assembled from simple monomers and adopt complex folded structures through networks of stabilizing noncovalent interactions. These interactions define secondary and tertiary structure and in most cases specify a unique three-dimensional architecture. Individual secondary or tertiary structures can also associate with one another to form multi-subunit quaternary structures. Nonnatural folded polymers have potential for similar structural versatility. Here we describe a pair of beta3-peptides whose sequences were designed to promote a 14-helix structure in water, favor hetero-oligomer formation, and disfavor nonspecific aggregation. These beta3-peptides assemble noncovalently into a well-defined hetero-oligomer characterized by a defined stoichiometry, a highly stabilized secondary structure, and a cooperative melting transition (TM > 55 degrees C). This work demonstrates that beta3-peptides can assemble into defined, cooperatively folded quaternary structures and constitutes an important step toward designing protein-like assemblies from nonnatural polymers.

Correlation of early orientational ordering of engineered λ 6–85 structure with kinetics and thermodynamics

Experimental and computational Φ-value analysis of two-state helical proteins has shown that definite interactions among helix-forming segments build up in the transition state ensemble, but this type of analysis is not applicable to downhill folders. Here, we ask whether orientational ordering of helix-forming segments occurs early on during folding of a downhill λ 6–85 mutant, and how much it correlates with the thermodynamics and kinetics of various λ 6–85 mutants that do have folding barriers. From a grand total of 5 μs of implicit solvent replica-exchange molecular dynamics, we conclude that under folding conditions segments 1 and 4 form more helical structure and orient correctly relative to the native structure more often than do segments 2 and 3. Helices 1 and 2 retain the most residual structure and orientation at high temperatures. This is further supported by experimental data showing that perturbations in helices 1 and 4 of this well-designed folder affect folding kinetics and stability more sensitively than elsewhere in the protein, and that the helix 1–2 only bundle retains a cooperative melting transition and helical CD spectrum. The correct orientational propensity of helices 1 and 4 at low temperature is in agreement with the work by Takada, Portman and Wolynes proposing initial structure formation during folding in helices 1 and 4 of the wild-type λ 6–85 protein, a two-state folder. Thus, the absence of a large barrier in the downhill mutant does not fundamentally alter the steps the wild-type protein takes to fold.

Raman study of the thermal behaviour and conformational stability of basic pancreatic trypsin inhibitor.

We have studied the thermal denaturation of native basic pancreatic trypsin inhibitor (BPTI) by monitoring the Raman bands in the 4000-400 cm(-1) range. In agreement with results obtained by calorimetry, a cooperative melting transition is observed starting at 75 degrees C. This transition is found to involve predominantly the unfolding of helical structures accompanied by beta-aggregation, loss of hydrophobic interactions between side chains and changes in CSSC dihedral angles. However, salt bridge breaking starts near 40 degrees C, as deduced from the nu(s)(COO(-)) band and from the bands close to 1320 and 1345 cm(-1) which for the first time have been shown to be due largely to vibrations of the arginine guanidyl group in BPTI. The thermal stability is, hence, attributable to cooperative contributions from hydrophobic and backbone hydrogen bond interactions as well as from disulfide bonds.

A calorimetric study of the folding-unfolding of an α-helix with covalently closed N and C-terminal loops

The thermal melting of a dicyclic 29-residue peptide, having helix-stabilizing side-chain to side-chain covalent links at each terminal, has been studied by circular dichroism spectropolarimetry (CD) and differential scanning calorimetry (DSC). The CD spectra for this dicyclic peptide indicate that it is monomeric, almost fully α-helical at −10 °C, and undergoes a reversible transition from the folded to the disordered state with increasing temperature. The temperature dependencies of the ellipticity at 222 nm and the excess heat capacity measured calorimetrically are well fit by a two-state model, which indicates a cooperative melting transition that is complete within the temperature ranges of these experiments (from −10 °C to 100 °C). This allows a complete analysis of the thermodynamics of helix formation. The helix unfolding is found to proceed with a small positive heat-capacity increment, consistent with the solvation of some non-polar groups upon helix unfolding. It follows that the hydrogen bonds are not the only factors responsible for the formation of the α-helix, and that hydrophobic interactions are also playing a role in its stabilization. At 30 °C, the calorimetric enthalpy and entropy values are estimated to be 650(±50) cal mol −1 and 2.0(±0.2) cal K −1mole −1, respectively, per residue of this peptide. Comparison with the thermodynamic characteristics obtained for the unfolding of double-stranded α-helical coiled-coils shows that at that temperature the enthalpic contribution of non-polar groups to the stabilization of the α-helix is insignificant and the estimated transition enthalpy can be assigned to the hydrogen bonds. With increasing temperature, the increasing magnitude of the negative enthalpy of hydration of the exposed polar groups should decrease the helix-stabilizing enthalpy of the backbone hydrogen bonds. However, the helix-stabilizing negative entropy of hydration of these groups should also increase in magnitude with increasing temperature, offsetting this effect.

An FTIR Study of the Complex Melting Behavior of α-Lactalbumin

We have studied the thermal denaturation of holo α-lactalbumin (α-LA) using FTIR spectroscopy by monitoring the absorbance of protein bands in the secondary structure sensitive amide I region. In agreement with results obtained by other techniques, a major cooperative melting transition is observed between 60 and 80 °C. This main transition is found to involve predominantly the unfolding of helical structures. Addition of excess Ca2+ ions raises the Tm of the cooperative melting transition but does not change the protein substructures involved in the transition. In addition to the main transition, there are at least two other distinct melting processes. One of them is gradual, is independent of excess Ca2+, and occurs throughout the measured temperature range below the major cooperative transition. This melting process involves denaturation of α-LA's β-hairpin. The other melting process is affected by the addition of excess Ca2+ and involves the denaturation of helical structures. In the absence of excess calcium, this transition, occurring between 45 and 60 °C, strongly overlaps with the major cooperative melting. Our results indicate that the β-hairpin of α-LA is not directly stabilized by the bound calcium ion present in the holo protein and melts independently from the rest of the protein.

Analysis of the Structure and Stability of a Backbone-Modified Oligonucleotide: Implications for Avoiding Product Inhibition in Catalytic Template-Directed Synthesis

The structural and thermodynamic origins of the destabilization of a backbone-modified DNA duplex 1, formed between d(CpGpTNTpGpC), containing a single aminoethyl group (−CH2−CH2−NH2+−) in place of the phosphodiester (−O−PO2-−O−) linkage of the central TT dimer, and d(GpCpApApCpG) are investigated. Analyses for the corresponding native duplex and two other related structural analogues of duplex 1 have been compared. Duplex 1 shows a cooperative thermal melting transition that is consistent with a two-state process. At a 2 mM concentration, the melting temperature of duplex 1 is reduced by 17 °C from the native duplex, and this decrease in stability is further assigned to an unfavorable decrease in enthalpy of 7 kcal mol-1 and a favorable increase in entropy of 15 eu mol-1. NMR structural analysis shows that the modified duplex 1 still adopts a canonical B-DNA conformation with Watson−Crick base pairing preserved; however, the CH2 group that replaces the native PO2- group in the modified backbone is flexib...

Collagen-Based Structures Containing the Peptoid Residue N-Isobutylglycine (Nleu): Conformational Analysis of Gly-Nleu-Pro Sequences by 1H-NMR and Molecular Modeling

Molecular modeling and 1H-NMR were employed to study the structure and stability of collagen-like triple helices composed of Gly-Nleu-Pro repeats. The compounds studied include the acetyl analogs Ac-(Gly-Nleu-Pro)n-NH2 (where n = 1, 3, 6, and 10) and the KTA conjugates KTA-[Gly-(Gly-Nleu-Pro)n-NH2]3 (where n = 3 and 6 and KTA denotes the Kemp triacid). The presence of collagen-like assembled structures is supported by a consistent set of experimental observations, which include the appearance of a distinct set of resonances, low hydrogen-exchange rates for Gly NH, cooperative melting transition, and observation of several interchain NOEs. Using 1H-NMR, the triple helicity was monitored as a function of chain length, template, and temperature. These studies show that (Gly-Nleu-Pro)n sequences have a somewhat higher triple-helical propensity than (Gly-Pro-Nleu)n sequences. In addition, our investigations have shown that unlike the triple helices composed of Gly-Pro-Nleu repeats those composed of Gly-Nleu-Pro repeats can access conformations in which the Nleu side chains are arrayed between Pro residues belonging to different triple-helix cross sections. These structural features may serve as a basis for free energy computations and for the study of higher-order structures such as collagen-like fibrils containing peptoid moities.

Collagen-Based Structures Containing the Peptoid Residue N-Isobutylglycine (Nleu). 6. Conformational Analysis of Gly-Pro-Nleu Sequences by 1H NMR, CD, and Molecular Modeling

Molecular modeling, 1H NMR, and CD were employed to study the structure and stability of collagen-like triple helices composed of Gly-Pro-Nleu repeats. The compounds studied include the acetyl analogs Ac-(Gly-Pro-Nleu)n-NH2 (where n = 1, 6, 9) and the KTA conjugates KTA-[Gly-(Gly-Pro-Nleu)n-NH2]3 (where n = 1, 3, 6, 9 and KTA denotes the Kemp triacid). The presence of collagen-like assembled structures was supported by a consistent set of experimental observations, including the appearance of a distinct set of resonances, low hydrogen exchange rates for Gly NH, KTA signal splitting, cooperative melting transition, and analysis of NOESY cross peaks. In this regard, the concept of ensemble interchain NOEs was introduced and used to establish the close packing of Gly, Pro, and Nleu residues in triple helices composed of Gly-Pro-Nleu repeats. In addition, the ensemble interchain NOEs gave insight into the puckering of the Pro ring and the conformations accessible to the Nleu side chain. The effect of the KTA template on triple helicity was studied and shown to consist in a net gain in the free energy of triple-helix formation, as also seen for Gly-Pro-Hyp sequences. This free energy gain led to the induction of an assembled collagen-like structure in the KTA conjugate containing six Gly-Pro-Nleu repeats per chain and to an increase in thermal stability of the compound containing nine Gly-Pro-Nleu repeats per chain.

Recognition of Single-Stranded Nucleic Acids by Triplex Formation: The Binding of Pyrimidine-Rich Sequences

Recognition of Single-Stranded Nucleic Acids by Triplex Formation: The Binding of Pyrimidine-Rich Sequences

Synthesis, characterization and solution properties of ras sequences modified by arylamine carcinogens at the first base of codon 61

The complementary pentadecamers d(5'-TACTCTTCTTGACCT) (strand A) and d(5'-AGGTCAAGAAGAGTA) (strand B), which span a portion of the mouse c-Ha-ras protooncogene centered around codon 61, were synthesized by using standard beta-cyanoethyl phosphoramidite chemistry and characterized by sequence analysis. Strand A, containing a sole guanine at the position corresponding to the first base of codon 61, was modified with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene or its 4-aminobiphenyl analogue. In both cases only the corresponding N-(deoxyguanosin-8-yl)arylamine adduct was formed, as judged from HPLC and UV analyses conducted after enzymatic hydrolysis of the modified oligomers. Nonmodified and modified pentadecamers were annealed with strand B. Cooperative melting transitions were observed with all samples, thus indicating the formation of stable duplexes. Melting temperatures decreased in the order nonmodified duplex greater than 2-aminofluorene-modified duplex greater than 4-aminobiphenyl-modified duplex, which indicated destabilization of the helical structure upon incorporation of the adducts, with 4-aminobiphenyl having the greatest effect. Circular dichroism spectra of all duplexes were characteristic of an overall right-handed B-type conformation, with no major conformational differences being detected between the two arylamine-modified oligomers.