Thermal Melting Profiles Research Articles

Computational and Biophysical Approaches to Identify Cell Wall-Associated Modulators in Salmonella enterica serovar Typhi.

An increase in the number of antibiotic-resistant bacterial pathogens, in recent times, has posed a great challenge for treating the affected patients. This has paved the way for the development and design of antibiotics against the previously less explored newer targets. Among these, peptidoglycan (PG) biosynthesis serves as a promising target for the design and development of novel drugs. The peptidoglycan cell wall synthesis in bacteria is essential for its viability. The enzyme class, Mur ligases, plays a key role in PG biosynthesis. Therefore, compounds with the ability to inhibit these enzymes (Mur ligase) can serve as potential candidates for developing small modulators. The enzyme, UDP-N-acetyl pyruvyl-glucosamine reductase(MurB), is essential for PG biosynthesis, a crucial part of the bacterial cell wall. The development of novel drugs to treat infections may thus focus on inhibiting MurB function. Understanding the mechanism of action of Mur B is central to developing efficient inhibitors. For the treatment of S. typhi infections, it is also critical to find therapeutic drugs that specifically target MurB. The enzyme Mur B from Salmonella enterica serovar Typhi (stMurB) was expressed and purified for biophysical characterization to gauge the molecular interactions and estimate thermodynamic stability, for determining attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism (CD) and validated by performing differential scanning calorimetry (DSC). An in silico virtual screening of various natural inhibitors was conducted with modelled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) obtained from in silico screening were validated for complex stability through molecular dynamics (MD) simulation. Further, fluorescence binding studies were undertaken for the selected natural inhibitors with stMurB alone and with its NADPH-bound form. The natural inhibitors, scopoletin and berberine, displayed lesser binding to stMurB compared to quercetin. Also, a stronger binding affinity was exhibited between quercetin and stMurB compared to NADPH and stMurB. Based on the above two findings, quercetin can be developed as an inhibitor of stMurB enzyme.

Interaction of isoquinoline alkaloids with pyrimidine motif triplex DNA by mass spectrometry and spectroscopies reveals diverse mechanisms

Isoquinoline alkaloids represent an important class of molecules due to their broad range of pharmacology and clinical utility. Prospective development and use of these alkaloids as effective anticancer agents have elicited great interest. In this study, in order to reveal structure-activity relationship, we present the characterization of bioactive isoquinoline alkaloid-DNA triplex interactions, with particular emphasis on the sequence selectivity and preference of binding to the two types of DNA triplexes, by electrospray ionization mass spectrometry (ESI-MS) and various spectroscopic techniques. The six alkaloids, including coptisine, columbamine, epiberberine, berberrubine, jateorhizine, and fangchinoline, were selected to explore their interactions with the TC and TTT triplex DNA structures. Berberrubine, fangchinoline, coptisine, columbamine, and epiberberine have preference for TC rich DNA sequences compared to TTT rich DNA triplex based on affinity values in MS. The experimental results from different fragmentation modes in tandem MS, subtractive and hyperchromic effects in UV absorption spectra, fluorescence quenching and enhancement in fluorescence spectra, and strong conformational changes in circular dichroism (CD) hinted that the interaction between isoquinoline alkaloid-TC/TTT DNA had diverse mechanisms including at least two different binding modes: the electrostatic binding and the intercalation binding. Interestingly, columbamine, berberrubine, and fangchinoline can stabilize TTT triplex as inferred from optical thermal melting profiles, while it was not the case in TC triplex. These results provide new insights into binding of isoquinoline alkaloids to pyrimidine motif triplex DNA.

Identification of natural small molecule modulators of MurB from Salmonella enterica serovar Typhi Ty2 strain using computational and biophysical approaches.

The increase of antibiotic-resistant bacterial pathogens has created challenges in treatment and warranted the design of antibiotics against comparatively less exploited targets. The peptidoglycan (PG) biosynthesis delineates unique pathways for the design and development of a novel class of drugs. Mur ligases are an essential component of bacterial cell wall synthesis that play a pivotal role in PG biosynthesis to maintain internal osmotic pressure and cell shape. Inhibition of these enzymes can interrupt bacterial replication and hence, form attractive targets for drug discovery. In the present work, we focused on the PG biosynthesis pathway enzyme, UDP-N-acetylpyruvylglucosamine reductase, from Salmonella enterica serovar Typhi (stMurB). Biophysical characterization of purified StMurB was performed to gauge the molecular interactions and estimate thermodynamic stability for determination of attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism and validated through differential scanning calorimetry experiment. Frequently used chemical denaturants, GdmCl and urea, were employed to study the chemical-induced denaturation of stMurB. In the search for natural compound-based inhibitors, against this important drug target, an in silico virtual screening based investigation was conducted with modeled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) returned were validated for complex stability through molecular dynamics simulation. Further, fluorescence binding studies were undertaken for the selected natural compounds with stMurB alone and with NADPH bound form. The compounds scopoletin and berberine, displayed lesser binding to stMurB whereas quercetin exhibited stronger binding affinity than NADPH. This study suggests that quercetin can be evolved as an inhibitor of stMurB enzyme.

Binding properties of pyrene-porphyrin dyad to G-quadruplexes in the presence of K+ and Na+ ion and their effect on stability

The binding property of 1-pyrenyl-tris(N-methyl-p-pyridino)porphyrin (a pyrene-porphyrin dyad; Py-TMPyP) to various G-quadruplexes formed from 5′-G3TnG3TGTG3TnG3 (n = 2 and 3, designated as S1 and S2, respectively) and conformation-dependent stability of the Py-TMPyP–quadruplex complexes were investigated via thermal melting profiles and circular dichroism (CD) spectral analyses. Upon binding to parallel (in the presence of K+) and antiparallel (in the presence of Na+) S1 and S2 quadruplexes, a bisignate CD signal in the Soret region was induced for all four G-quadruplexes, indicating that the direction of interaction of the electric transition moments of the porphyrins differed. This suggested that the porphyrin binding site in the G-quadruplex is highly unsymmetric. Py-TMPyP induced a moderately negative CD signal in the pyrene absorption region when binding to the parallel S1 quadruplex and parallel/antiparallel mixed S2 quadruplex. In contrast, a strongly negative CD signal was observed for the Py-TMPyP bound to antiparallel S1 quadruplex, while no CD signal appeared for the antiparallel S2 quadruplex. Considering that two and three thymine bases are present in the loops of S1 and S2, respectively, the number of the thymines in the loop is important for the interaction of pyrene and the G-quadruplex. The thermal melting study showed that the antiparallel form of the G-quadruplex was generally less stable compared to the corresponding parallel conformation. The presence of Py-TMPyP caused destabilization of the G-quadruplexes.

Impact of Phosphorylation on MDH2 Structure, Function and Protein

Mitochondrial malate dehydrogenase (MDH2) is an enzyme utilizing the NAD/NADH cofactor system in the citric acid cycle to reversibly oxidize malate to oxaloacetate, where citrate synthase (CS) uses oxaloacetate to produce citrate. Coupling of the MDH2‐CS of the two reactions is critical as the thermodynamics of MDH2 alone would not drive the cycle forward. However, shuttling of carbon through the cycle during gluconeogenesis necessitates an uncoupling of the two proteins, allowing for the reversal of the reaction. Thus, a mechanism that favors the known interaction and disrupts substrate channeling through these two enzymes must exist. We performed an analysis of putative MDH2 phosphorylation sites, identified the potential kinases, and conducted a meta‐analysis of mass spectrometry data bases of phosphorylation. We have identified 13 predicted, and 30 identified phosphorylated sites of mammalian MDH2. Further analysis of possible interaction sites and mapping functional domains with these phosphorylation sites highlight the important yet unknown role of phosphorylation to regulate MDH structure and function. In order to better understand MDH2‐CS interactions and MDH function, we synthesized four phosphomimics (S/T‐D) using site directed mutagenesis on a codon optimized human MDH2 gene in a His‐tagged, pET28a construct for bacterial expression. Using purified protein, we show impact of each mutation structure with circular dichroism and the thermal melting profile of the phosphomimics and the wildtype hMDH2. The impact of phosphorylation on MHD function was also studied by comparing kinetic values. Lastly the impact of the mutations on MDH‐CS binding was analyzed using a micro‐protein thermal shift assay.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932

Not all pycnodysostosis-related mutants of human cathepsin K are inactive - crystal structure and biochemical studies of an active mutant I249T.

Human cathepsin K (CTSK) is a collagenolytic lysosomal cysteine protease that plays an important role in bone turnover. Mutation in CTSK gene is associated with loss of collagenolytic activity of CTSK leading to an autosomal recessive bone disorder called pycnodysostosis. Although a number of pycnodysostotic missense mutations have been reported, underlying mechanism of the disease is not clear. In this study, we investigated invitro six recombinant pycnodysostosis-related mutants of human CTSK (G79E, I249T, G243E, G303E, G319C and Q187P). While all the mutants, like wild-type, show similar high levels of expression in Escherichiacoli, four of them (G79E, G303E, G319C and Q187P) are inactive, unstable and spontaneously degrade during purification process. In contrast, proteolytic/collagenolytic activity, zymogen activation kinetics and stability of G243E and I249T mutants are nominally affected. Crystal structure of I249T at 1.92Å resolution shows that the mutation in R-domain causes conformational changes of a surface loop in the L-domain although the catalytic cleft remains unaltered. Molecular simulation, normal mode analysis and fluorescence lifetime measurement eliminated the possibility that the change in L-domain surface loop orientation is a crystallization artefact. CD-based thermal melting profile indicates that stability of I249T is significantly higher than wild-type. Our studies first time reports that pycnodysostosis-related mutations do not always lead to complete loss of general proteolytic activity or specific collagenolytic activity of CTSK. The first crystal structure of a pycnodysostotic mutant (I249T) provides critical information that may pave new avenues towards understanding the disease at molecular level. DATABASE: The atomic co-ordinates and structure factors for I249T mutant of human CTSK (codes 5Z5O) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Rapid solid-phase syntheses of a peptidic-aminoglycoside library

A library of mono- and di-amino acid peptidic-aminoglycosides (PAs), with kanamycin and neomycin as the model aminoglycosides, was systematically and rapidly synthesized via solid phase peptide synthesis. Aminoglycosides were first converted into N-Boc protected carboxylic acids and fifteen l-amino acids were then used in the diversification of the full library. The approach outlined describes a rapid synthetic procedure where >200 PA compounds can be synthesized in a few months with 85–95% purity. UV thermal denaturation assessed the binding stabilization by PAs to model human and bacterial A-site rRNA sequences. Significant differences were found in thermal melting profiles among PAs that were attributed to specific amino acid sequences. Neomycin PAs lead to a much larger variation in the stabilization of A-site rRNA sequences (ΔTm = 2.6–17.1 °C) as compared to kanamycin PAs (ΔTm = 0.4–4.3 °C). Kanamycin PAs had little activity against Gram-negative and Gram-positive bacteria as compared with neomycin PAs that had significant antibacterial activity with MIC ranging from 2 to 16 μM.

Studies on DNA Interaction with 2(3),9(10), 16(17), 23(24)‐Tetrakis 2((4‐Phenoxyphenyl) Diazeylbenzoic acid)phthalocyaninato Zinc (II) Compound

AbstractIn this study, 2(3), 9(10), 16(17), 23(24)‐tetrakis 2((4‐phenoxyphenyl) diazeylbenzoic acid)phthalocyaninato zinc (II) compound was chosen to study DNA binding activities. The binding activities of zinc (II) phthalocyanine compound bearing 2(3),9(10), 16(17), 23(24)‐tetrakis 2((4‐Phenoxyphenyl) diazeylbenzoic acid) ligand with Calf Thymus (CT)‐DNA was investigated using absorption titration, fluorescence spectroscopy, cyclic voltammetry and thermal melting profile in a Tris‐HCl buffer system at pH 7.0. In addition to these methods, gel electrophoresis and viscosity measurements were also carried out to determine DNA binding activities with compound 6 in a buffer solution. The results indicated that compound 6 interacts with CT‐DNA through partial intercalation binding mode.

Molecular and Thermodynamic Studies on DNA Triplex Formed in the Promoter Region of HMGB1 Gene as a Selective Target for Anticancer Drugs.

HMGB1 (High Mobility Group Box-1) is a very versatile highly abundant architectural protein that plays multiple roles in human health and diseases. Under physiological condition it serves as an amazing assortment of roles in different compartments of cell. The reported high expression of HMGB1 in almost all types of human cancers and inflammatory diseases makes it a critical molecular therapeutic target. In the present study, we have mobilized a proximal twenty one base pair nucleotide (21RY) which is in the promoter region (-55 to-75) of hmgb1 gene and targeted it with triplex forming oligonucleotide (TFO) in combination with two widely used chemotherapeutic drugs, actinomycin (ACT) and adriamycin (ADM). The interaction of actinomycin and adriamycin to 21R*R•Y DNA triplex was studied using UV melting profiles, CD spectroscopy, spectrofluorimetry and Isothermal titration calorimetry. The 21R*R•Y formation was confirmed from biphasic thermal melting profiles, continuous variation method, analysis of CD marker band and thermodynamic parameters. The binding of ADM and ACT to 21R*R•Y was characterized by hypochromic and bathochromic shift in their respective absorption spectrum, quenching (ADM) and enhanced fluorescence (ACT) of steady-state fluorescence intensity, perturbation in the circular dichroic spectrum and change in thermal melting temperatures. The ITC profile and Scatchard plot analysis indicate non-cooperative and higher binding affinity of these drugs to 21R*R•Y compared to their corresponding duplexes. Therefore, combining these chemotherapeutic drugs with triplex forming oligonucleotide may offer new diagnostic and therapeutic options in targeting a gene of interest more specifically with fewer side effects. This study shows that ACT and ADM effectively recognize 21R*R•Y triplex DNA formed on the hmgb1 promoter region.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV–visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103M−1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Third strand stabilization of poly(U)·poly(A)* poly(U) triplex by the naturally occurring flavone luteolin: A multi-spectroscopic approach

Naturally occurring flavonoid luteolin (LTN) was found to interact with double stranded poly(A).poly(U) and triple stranded poly(U)·poly(A)*poly(U) with association constants of the order of 104M−1. The association was monitored by various spectroscopic and viscometric techniques. Non-cooperative binding was observed for the association of LTN with two different polymorphic forms of RNA. Intercalation mode of binding was confirmed by fluorescence quenching and viscometric experiments. Thermal melting profiles indicated greater stabilization of the Hoogsteen base paired third strand (∼16°C) compared to Watson-Crick double strand (∼5°C) of RNA by LTN. Since the interaction of naturally occurring small molecules with RNA is an active area of research, this study has led to great openings to explore LTN as RNA targeted therapeutic agent.

Behavior of the Guanine Base in G‐quadruplexes Probed by the Fluorescent Guanine Analog, 6‐Methyl Isoxanthopterin

In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G‐stem of the G‐quadruplex formed from the G3T2G3TGTG3T2G3 sequence (G‐quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6‐methyl isoxanthopterin (6MI) (G‐quadruplex 2 and 3, respectively). For all G‐quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G‐quadruplex. The fluorescence intensity of G‐quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G‐quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G‐quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G‐quadruplex 3 was proposed.

Spectroscopic Studies of the Binding Interactions of Phenothiazininum Dyes (Thionine Acetate, Azure A and Azure B) with Calf-thymus DNA

The double helical structure of DNA offers various binding sites for the interaction of ligands or proteins. Interactions using minor groove, major groove, and through intercalation are the major types of binding mechanisms of DNA-ligand interactions. The lowering in the absorption intensity along with bathochromic shift is the indication of intercalation binding mode of the dye into the base pairs of the DNA. In this study, the interaction of phenothiazine dyes with calf-thymus DNA (ctDNA) in physiological buffer (pH 7.4) was studied using UV-visible, fluorescence, circular dichroism (CD), and UV-thermal denaturation spectroscopy. The binding constants were calculated at different temperatures with the help of fluorescence spectroscopy. CD signals signify that B-form of DNA might become more compact, upon binding of the dyes. Also, induced circular dichroism is observed which confirms the dye-DNA complex formation. Stabilization of DNA double helix upon binding with dyes was confirmed by the increase in Tm of ctDNA. Based on thermal melting profiles, it was found that thionine acetate is most promising in stabilizing the DNA double helix, in comparison to other two dyes. Also, binding constants calculated by fluorescence is in accordance with the thermal melting analysis. These results are indicative of the intercalation binding mode between dyes and the DNA. The binding affinity of the dyes to DNA is found to be in order as thionine acetate > azure A > azure B. Such preliminary studies facilitate our understanding about various types of DNAligand interactions and provide clues for designing new and more effective drugs.

Differential Scanning Fluorimetry for Monitoring RNA Stability

Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA-binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand-induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single-stranded (or unstructured regions) and double-stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity.

Duplication of Genes in an ATP-binding Cassette Transport System Increases Dynamic Range While Maintaining Ligand Specificity

Many bacteria exist in a state of feast or famine where high nutrient availability leads to periods of growth followed by nutrient scarcity and growth stagnation. To adapt to the constantly changing nutrient flux, metabolite acquisition systems must be able to function over a broad range. This, however, creates difficulties as nutrient concentrations vary over many orders of magnitude, requiring metabolite acquisition systems to simultaneously balance ligand specificity and the dynamic range in which a response to a metabolite is elicited. Here we present how a gene duplication of a periplasmic binding protein in a mannose ATP-binding cassette transport system potentially resolves this dilemma through gene functionalization. Determination of ligand binding affinities and specificities of the gene duplicates with fluorescence and circular dichroism demonstrates that although the binding specificity is maintained the Kd values for the same ligand differ over three orders of magnitude. These results suggest that this metabolite acquisition system can transport ligand at both low and high environmental concentrations while preventing saturation with related and less preferentially metabolized compounds. The x-ray crystal structures of the β-mannose-bound proteins help clarify the structural basis of gene functionalization and reveal that affinity and specificity are potentially encoded in different regions of the binding site. These studies suggest a possible functional role and adaptive advantage for the presence of two periplasmic-binding proteins in ATP-binding cassette transport systems and a way bacteria can adapt to varying nutrient flux through functionalization of gene duplicates.

Molecular basis of thermal stability in truncated (2/2) hemoglobins

BackgroundUnderstanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. MethodsWe used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. ResultsThe results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. ConclusionsThis gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. General significanceThese results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.

Human DNA damage by the synergistic action of 4‐aminobiphenyl and nitric oxide: An immunochemical study

4-Aminobiphenyl (4-ABP), an aromatic amine is a major environmental carcinogen found mainly in cigarette smoke. It has been vastly implicated in mutagenesis and cancer development. In this study, commercially available human placental DNA was exposed to 4-ABP (1.3 mM) in presence of sodium nitroprusside (SNP; 8 mM) at 37°C for 3 h. The 4-ABP + SNP-mediated structural changes in human DNA were studied by ultraviolet, circular dichroism and fluorescence spectroscopy, thermal melting profile, agarose gel electrophoresis, and nuclease S1 digestibility assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Modification was also visualized in agarose gel electrophoresis. Furthermore, nuclease S1 digestibility confirmed the generation of single strand breaks. Rabbits challenged with 4-ABP-SNP-modified human DNA-induced high-titer immunogen-specific antibodies, which showed Cross-reaction with modified/unmodified DNA bases and ss-DNA in competitive inhibition assay. The immunogen specificity of induced antibodies against 4-ABP-SNP-modified human DNA was further confirmed in gel retardation assay. It may be concluded that induction of anti-modified DNA antibodies could be due to perturbation in the DNA structure and its subsequent recognition by immunoregulatory cells as a foreign molecule.

Analysis of Human DNA-Arginine Photoadduct Modified with Peroxynitrite

The aim of the study is the biochemical characterization of human DNA modified with arginine and peroxynitrite. In the present study, DNA was isolated from human blood cells and its adduct was formed with one of the amino acid, arginine. The DNA-arginine adduct was then modified with peroxynitrite, a reactive nitrogen species. The modified DNA adduct was characterized by ultraviolet (UV) absorption spectroscopy, thermal melting profile, and electrophoresis studies. UV spectroscopic analysis of the photoadduct showed hyperchromicity, indicating the formation of single-strand breaks and photomodification. Thermal denaturation studies of DNA-arginine adduct and peroxynitrite-modified adduct showed a decrease in the temperature (T m) value by 4.5°C and an increase in the T m of 8°C, respectively. Peroxynitrite modification is evident by an increase in the T m value and a change in the migration pattern of native and modified photoadducts on agarose gel electrophoresis. The DNA-arginine and peroxynitrite-modified photoadducts could have important implications in various pathophysiological and immunopathological conditions.

Structural properties of DNA oligomers containing (GACX)n and (GAXC)n tandem repeats

The antisense DNA sequence of mature mouse micro RNA, miR341, includes three repeats of the tetranucleotide (GACC). The -GAC- repeat is known to form a parallel duplex, in acidic environments. The thermal melting profile of miR341 DNA, at pH 4, 5, and 6 indicates the formation of a very stable structure, which loses its stability when pH is increased. Thus, the addition of a cytosine at the 3' end of the (GAC) motif preserves the molecule's potential to fold into an unusual structure at low pH. The effect of modifying the nucleotide composition of the GACC sequence on the secondary structures formed by oligomers containing seven tandem repeats of the altered motifs was examined here. UV melting profiles were determined, as a function of pH, for 28-mers of the two series (GAXC)(7) and (GACX)(7) (X= A/C/T/G)(.) The sequence (GACC)(7) was found to be extremely sensitive to pH variations, with a stable structure formed at pH 5 (T(m) ≥ 60°C). NMR spectroscopy established that the low pH structure is not B-DNA. (GACA)(7) and (GACT)(7) also formed stable structures at low pH but the addition of guanine at the 3'end, as seen in the (GACG) series resulted in the loss of this property. Introducing a break in the 5'-GAC-3' motif, explored in the (GAXC) series, also inhibits formation of stable structures under acidic conditions.

Protoberberine Alkaloids Berberine, Palmatine, and Coralyne Binding to Poly(dT)⋅(Poly(dA)⋅Poly(dT)) Triplex: Comparative Structural Aspects and Energetics Profiles of the Interaction

AbstractThe interaction of bioactive protoberberine alkaloids berberine, palmatine, and coralyne with the DNA triplex poly(dT)⋅(poly(dA)⋅poly(dT)) was studied using biophysical and calorimetric techniques. All three alkaloids bound the triplex cooperatively. Berberine and palmatine predominantly stabilized the triplex structure, while coralyne stabilized both triplex and duplex structures as inferred from optical thermal melting profiles. Fluorescence quenching, polarization, and viscometric studies hinted at an intercalative mode of binding for the alkaloids to the triplex, coralyne being more strongly intercalated compared to partial intercalation of berberine and palmatine. The overall affinity of coralyne was two order higher (2.29×107 M−1) than that of berberine (3.43×105 M−1) and palmatine (2.34×105 M−1). Isothermal titration calorimetric studies revealed that the binding to the triplex was favored by negative enthalpy change (ΔH=−3.34 kcal/mol) with favorable entropy contribution (TΔS = 4.07 kcal/mol) for berberine, favored by almost equal negative enthalpy (ΔH =−3.88 kcal/mol) and entropy changes (TΔS = 3.37 kcal/mol) for palmatine, but driven by large enthalpy contributions (ΔH =−25.62 kcal/mol and TΔS =−15.21 kcal/mol) for coralyne. These results provide new insights on the binding of isoquinoline alkaloids to the DNA triplex structure.