Synthetic Polynucleotides Research Articles

SPECTROPHOTOMETRIC STUDY OF COMPLEXES OF SOME INTERCALATORS WITH DOUBLE-STRANDED AND SINGLE-STRANDED NUCLEIC ACIDS

Interaction of ethidium bromide (EtBr), acridine orange (AO) and non-intercalator methylene blue (MB) with DNA as well as interaction of EtBr and MB with single-stranded synthetic polynucleotides poly(rA) and poly(rU) have been studied. It was revealed that in absorption spectra of EtBr and MB with DNA or single-stranded polynucleotides a real- or pseudo-isosbestic point appears. Particularly, in absorption spectra of the complexes of classical intercalator EtBr with DNA the real isosbestic point is formed, but in the case of non-classical intercalator MB a pseudo-isosbestic point is formed, while in the absorption spectra of their complexes with single-stranded polynucleotides a pseudo-isosbestic point is formed. It was found out that for another classical intercalator AO there is no a real or pseudo-isosbestic point.

Study of complexation of single-stranded poly(rA) and poly(rU) with methylene blue

To reveal the effect of DNA- or RNA-specific low-molecular compounds on cellular processes on the molecular level, we have carried out the studies with the application of spectroscopic methods. It is necessary for the understanding of structural-functional properties of nucleic acids in cell. In this work the interaction of DNA-specific thiazine dye methylene blue (MB) with synthetic polynucleotides poly(rA) and poly(rU) was studied. The interaction of MB with synthetic polyribonucleotides poly(rA) and poly(rU) was examined in the solution with high ionic strength in a wide phosphate-to-dye (P/D) range, using the absorption and fluorescence spectroscopies, as well as the fluorescence 2D spectra and 3D spectra analyses were given. Values of the fluorescence quenching constants for the complexes of MB with poly(rA) and poly(rU) were calculated (KSV is the Stern-Volmer quenching constant). Two different modes of MB binding to single-stranded (ss-) poly(rA) and poly(rU) and to their hybrid double-stranded (ds-) structure – poly(rA)-poly(rU) were identified. This ligand binds to ss-poly(rA) and poly(rA)-poly(rU) by semi-intercalation and electrostatic modes, but to ss-poly(rU) the prevailing mode is the electrostatic interaction. Communicated by Ramaswamy H. Sarma

Small-Molecule Natural Product Physachenolide C Potentiates Immunotherapy Efficacy by Targeting BET Proteins.

Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand-dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8-dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. SIGNIFICANCE: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3374/F1.large.jpg.

Thermodynamics of the Interaction of Synthetic Polynucleotide Poly(rA)-Poly(rU) with Intercalators

The melting of poly(rA)-poly(rU) and its complexes with the intercalators — methylene blue (MB) and ethidium bromide (EtBr) has been carried out at the ionic strength of the solution 0.02, 0.04, and 0.1 M. It was revealed that this polynucleotide has the non-stable double-stranded structure at the ionic strength of 0.02 M and it becomes more stable at the complex-formation with the mentioned ligands. The increase of the solution ionic strength also results in the stabilization of the double-stranded structure of the polynucleotide, in consequence of which the interaction of the MB and EtBr with the poly(rA)-poly(rU) becomes more preferable and more beneficial thermodynamically. It was shown that at the ionic strength of 0.1 M the poly(rA)-poly(rU) takes a structure that is more available to the intercalation of both ligands. In the case of the MB the main binding mode becomes a semi-intercalation, while in the case of the EtBr all the intrinsic binding modes are displayed. It was also shown that in the case of the EtBr the values of ΔH and ΔS increase, while in the case of the MB, ΔH and ΔS those values increase, then decrease at the enhancement of the ionic strength of the solution.

The Immunogenicity in Mice of HCV Core Delivered as DNA Is Modulated by Its Capacity to Induce Oxidative Stress and Oxidative Stress Response

HCV core is an attractive HCV vaccine target, however, clinical or preclinical trials of core-based vaccines showed little success. We aimed to delineate what restricts its immunogenicity and improve immunogenic performance in mice. We designed plasmids encoding full-length HCV 1b core and its variants truncated after amino acids (aa) 60, 98, 152, 173, or up to aa 36 using virus-derived or synthetic polynucleotides (core191/60/98/152/173/36_191v or core152s DNA, respectively). We assessed their level of expression, route of degradation, ability to trigger the production of reactive oxygen species/ROS, and to activate the components of the Nrf2/ARE antioxidant defense pathway heme oxygenase 1/HO-1 and NAD(P)H: quinone oxidoreductase/Nqo-1. All core variants with the intact N-terminus induced production of ROS, and up-regulated expression of HO-1 and Nqo-1. The capacity of core variants to induce ROS and up-regulate HO-1 and Nqo-1 expression predetermined their immunogenicity in DNA-immunized BALB/c and C57BL/6 mice. The most immunogenic was core 152s, expressed at a modest level and inducing moderate oxidative stress and oxidative stress response. Thus, immunogenicity of HCV core is shaped by its ability to induce ROS and oxidative stress response. These considerations are important in understanding the mechanisms of viral suppression of cellular immune response and in HCV vaccine design.

Spectroscopic and molecular docking investigation of polynucleotides and DNA binding affinity to Nile blue dye

ABSTRACTWe used UV-vis absorption spectroscopy, fluorescence spectrophotometry and molecular docking calculations to investigate intermolecular interaction between the cationic dye, Nile blue (NB), and synthetic polynucleotides, poly(A-T), poly(G-C) and calf thymus DNA (Ct-DNA) at physiological pH. Strong hypsochromic absorbance and fluorescence quenching were observed that showed strong binding of NB to these polynucleotides and DNA. The binding affinity values derived from maximum absorption of the spectra of NB bound to various polynucleotides and Ct-DNA concentrations suggests that NB exhibits greater binding affinity to poly(G-C) than to poly(A-T). The thermodynamic parameters suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of NB to DNA. The molecular docking results suggested that NB was an intercalator of the stacked base pairs of Ct-DNA.

Synchrotron-radiation vacuum-ultraviolet circular dichroism spectroscopy in structural biology: an overview.

Circular dichroism spectroscopy is widely used for analyzing the structures of chiral molecules, including biomolecules. Vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy using synchrotron radiation can extend the short-wavelength limit into the vacuum-ultraviolet region (down to ~160 nm) to provide detailed and new information about the structures of biomolecules in combination with theoretical analysis and bioinformatics. The VUVCD spectra of saccharides can detect the high-energy transitions of chromophores such as hydroxy and acetal groups, disclosing the contributions of inter- or intramolecular hydrogen bonds to the equilibrium configuration of monosaccharides in aqueous solution. The roles of hydration in the fluctuation of the dihedral angles of carboxyl and amino groups of amino acids can be clarified by comparing the observed VUVCD spectra with those calculated theoretically. The VUVCD spectra of proteins markedly improves the accuracy of predicting the contents and number of segments of the secondary structures, and their amino acid sequences when combined with bioinformatics, for not only native but also nonnative and membrane-bound proteins. The VUVCD spectra of nucleic acids confirm the contributions of the base composition and sequence to the conformation in comparative analyses of synthetic poly-nucleotides composed of selected bases. This review surveys these recent applications of synchrotron-radiation VUVCD spectroscopy in structural biology, covering saccharides, amino acids, proteins, and nucleic acids.

The Peculiarities of Charge Motion in the Molecular Polynucleotide Chains of Finite Length. The Rapid Formation of a Moving Polaron State

The numerical experiments which demonstrate the possibility of charge transfer in a homogeneous G/C DNA chain in the absence of an electric field have been carried out. As a model, which describes the dynamics of a DNA molecule, was considered the nonlinear Peyrard-Bishop-Dauxois-Holstein model. It is commonly supposed that the main electric current carrier in homogeneous synthetic polynucleotide chains is the polaron. We have previously studied the peculiarities of polaron motion in molecular polynucleotide chains of finite length. It was shown that a polaron placed at the initial moment of time not in the center of the chain acquires the ability to move in the absence of an electric field and in the absence of any additional excitations in the chain. The numerical experiments which demonstrate the possibility of polaron charge transfer in a homogeneous finite unclosed G/C DNA chain due to the interaction with localized excitations have been carried out in the absence of an electric field. In this study, at the initial moment of time, a polaron is not added to the chain, but a charge localized in the region of a certain number of neighboring sites displaced from the equilibrium positions. The motion of the charge in the chain is caused by choice of these specified initial conditions, which ensure the rapid formation of the polaron state and, as a consequence, charge transfer along the chain. For the assignment of the external nonlinear excitations, we used nonzero values of the displacements of particles and/or their velocities at the initial instant of time. Non-zero values of chain sites velocities at the initial time were used to stimulate the motion of the charge. It is shown that for the rapid formation of the polaron state, the initial conditions must correspond to the parameters of the polaron, which is formed in the chain under the chosen parameters. It is shown that, depending on the parameters of the chain and on the parameters of the selected initial conditions, the charge can be transferred along the chain over long distances.

New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA.

Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus, negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photoinitiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of six atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in 30 seconds, as characterized by size-exclusive chromatography (SEC) and infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

Fluorescent ZnO-Au Nanocomposite as a Probe for Elucidating Specificity in DNA Interaction.

In this work, we report the interaction of a fluorescent ZnO–Au nanocomposite with deoxyribonucleic acid (DNA), leading to AT-specific DNA interaction, which is hitherto not known. For this study, three natural double-stranded (ds) DNAs having different AT:GC compositions were chosen and a ZnO–Au nanocomposite has been synthesized by anchoring a glutathione-protected gold nanocluster on the surface of egg-shell-membrane (ESM)-based ZnO nanoparticles. The ESM-based bare ZnO nanoparticles did not show any selective interaction toward DNA, whereas intrinsic fluorescence of the ZnO–Au nanocomposite shows an appreciable blue shift (Δλmax = 18 nm) in the luminescence wavelength of 520 nm in the presence of ds calf thymus (CT) DNA over other studied DNAs. In addition, the interaction of the nanocomposite through fluorescence studies with single-stranded (ss) CT DNA, synthetic polynucleotides, and nucleobases/nucleotides (adenine, thymine, deoxythymidine monophosphate, deoxyadenosine monophosphate) was also undertaken to delineate the specificity in interaction. A minor blue shift (Δλmax = 5 nm) in the emission wavelength at 520 nm was observed for single-stranded CT DNA, suggesting the proficiency of the nanocomposite for discriminating ss and ds CT DNA. More importantly, fluorescence signals from the nano-bio-interaction could be measured directly without any modification of the target, which is the foremost advantage emanated from this study compared with other previous reports. The AT base-pair-induced enhancement was also found to be highest for the melting temperature of CT DNA (ΔTmCT = 6.7 °C). Furthermore, spectropolarimetric experiments followed by calorimetric analysis provided evidence for specificity in AT-rich DNA interaction. This study would lead to establish the fluorescent ZnO–Au nanocomposite as a probe for nanomaterial-based DNA-binding study, featuring its specific interaction toward AT-rich DNA.

Spectroscopic Study of the Binding of Netropsin and Hoechst 33258 to Nucleic Acids

The interaction of groove binding compounds — peptide antibiotic (polyamide) netropsin and fluorescent dye (bisbenzimidazole) Hoechst 33258 — with the double-stranded DNA and synthetic double-stranded polynucleotide poly(rA)-poly(rU) has been studied by spectrophotometry. Absorption spectra of these ligand complexes with nucleic acids have been obtained. Spectral changes at the complexation of individual ligands with the mentioned nucleic acids reveal the similarity of binding of each of these ligands with both DNA and RNA. Based on the spectroscopic measurements, the binding parameters of netropsin and Hoechst 33258 binding to DNA and poly(rA)-poly(rU) – K and n, as well as the thermodynamic parameters ΔS, ΔG, and ΔH have been determined. It was found that the binding of Hoechst 33258 to both nucleic acids is accompanied by a positive change in enthalpy, while in the case of netropsin the change in enthalpy is negative. Moreover, the contribution of entropy to the formation of the complexes is more pronounced in the case of Hoechst 33258.

Interaction of Methylene Blue with Synthetic Polynucleotide Poly(dA)-Poly(dT)

The interaction of methylene blue (MB) with poly(dA)-poly(dT) has been studied. It was revealed that MB binds to this polynucleotide by strong and weak modes. It was shown that the strong binding mode of MB is realized by semi-intercalation. Apart from the strong mode, this ligand binds to AT-sequences by weak electrostatic modes as well. Values of the binding constant [Formula: see text] and number of base pairs [Formula: see text] per binding site of MB for the mentioned modes were determined. It was shown that at the interaction with AT sequences MB does not behave itself as a groove binding compound.

Dependence of the base sequence on the [Cu(2,2′-bipyridine)2(NO3)](NO3)-induced oxidative DNA cleavage probed by linear dichroism

The oxidative DNA cleavage induced by the [Cu(2,2′-bipyridine)2(NO3)]NO3 (Cu(bpy)2) complex was examined using the linear dichroism (LD) technique. Using this method, the oxidative DNA cleavage by the Fenton mechanism was reported to occur through two sequential first-order reactions. As the Cu(bpy)2 concentration increased, the rate constant of both first order reactions increased, as expected. The activation energy of the second step was estimated to be 0.190 kJ mol−1. A similar method was applied for various synthetic polynucleotides. Poly[d(G-C)2], poly(dG)·poly(dC), and poly(dA)·poly(dT) produced a time-dependent decrease in LD, which could be elucidated by a single component exponential decay. This observation is in contrast to Fenton-type oxidative DNA cleavage. On the other hand, poly[d(A-T)2] produced a time-dependent decrease in the LD magnitude, which could be elucidated by two sequential first order reactions.

Abstract 2159: A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands

Abstract Recent studies have demonstrated a role of toll-like receptor 3 (TLR3) signaling for the initiation of apoptosis in some malignant cells. We have previously shown that, withanolide E (WE), a 17β-hydroxywithanolide (17-BHW) natural product derived from the medicinal plant Physalis peruviana was capable of sensitizing tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by reducing cellular levels of the anti-apoptotic protein cFLIP. Animal studies also revealed that WE sensitized human renal carcinoma cells to apoptosis at concentrations that did not promote apoptosis in normal cells. Thus we further screened a library of 30 natural and semi-synthetic 17-BHWs for their ability to promote death ligand-mediated cancer cell death. Among the 30 compounds tested, LG-02 (physachenolide C) was found to be 4-5 fold more potent than WE in sensitizing some human renal carcinoma and melanoma cells to apoptotic cell death in response not only to TRAIL but also to the synthetic polynucleotide poly (I:C), which is known to mimic anti-viral responses by activating TLR3 signaling. To date there are no withanolides known to have this dual apoptosis sensitizing activity. LG-02 and Poly (I:C) treatment resulted in increased activation of caspase-8, and apoptosis was blocked by the pan caspase inhibitor zVAD-FMK. Poly (I:C)-driven apoptosis signaling was dependent on endosomal acidification, but independent of IRF 3 and Interferon α/β signaling. Molecular studies suggested a role for changes in the anti-apoptotic proteins cFLIP, IAPs, and Livin on apoptosis signaling in LG-02 treated cells. Loss of cIAP activity is reported to promote spontaneous formation of an intracellular death-inducing protein platform the ripoptosome, that can activate either apoptosis or necroptosis. Immunoprecipitation of either the TRAIL death-inducing signaling complex (DISC) or the Poly (I:C) ripoptosome, demonstrated enhanced levels of FADD and RIP1 and decreased levels of cFLIP in these macromolecular apoptosis signaling complexes in LG-02 treated cells. Intratumor administration of LG-02 and Poly (I:C) in a xenograft M14 melanoma model provided therapeutic benefit leading to complete tumor regression in 90 % of the mice as compared to control mice. Further studies with active 17-BHWs could lead to the identification of more potent analogues, and novel and common therapeutic targets involved in apoptosis signaling in response to both TNF death receptor family members as well as TLR3 ligands.Funded by FNLCR Contract HHSN261200800001E Citation Format: Poonam Tewary, Alan D. Brooks, Ya-ming Xu, Kithsiri E.M Wijeratne, Leslie A. Gunatilaka, Thomas J. Sayers. A specific 17-beta-hydroxywithanolide (LG-02) sensitizes cancer cells to apoptosis in response to TRAIL and TLR3 ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2159. doi:10.1158/1538-7445.AM2017-2159

Selective Binding of Genomic Escherichia coli DNA with ZnO Leads to White Light Emission: A New Aspect of Nano-Bio Interaction and Interface.

Here, we report for the first time, a novel and intriguing application of deoxyribonucleic acid (DNA) in the area of optics by demonstrating white light emission by tuning the emission of a nanomaterial, ZnO rods, exhibiting surface defects, in the presence of genomic Escherichia coli DNA with a comparatively high quantum efficiency. In order to understand the DNA specificity, we have also studied the interaction of ZnO with CT, and ML DNA, ss EC DNA, synthetic polynucleotides and different mononucleosides and bases. Further, in order to understand the effect of particle shape and defects present in ZnO, we have also extended our study with ZnO rods prepared at higher temperature exhibiting red emission and ZnO particles exhibiting yellow emission. Interestingly, none of the above studies resulted in white light emission from ZnO-DNA complex. Our studies unequivocally confirmed that the concentration and the nature of DNA and ZnO together plays a crucial role in obtaining CIE coordinates (0.33, 0.33) close to white light. The much enhanced melting temperature (Tm) of EC DNA and the energetics factors confirm enhanced hydrogen bonding of ZnO with EC DNA leading to a new emission band. Our experimental observations not only confirm the selective binding of ZnO to EC DNA but also open a new perspective for developing energy saving light emitting materials through nano-bio interactions.

Ground and excited state interactions of metalloporphyrin PtTMPyP4 with polynucleotides [poly(dG-dC)]2 and [poly(dA-dT)

The ground- and excited-state interactions of Pt(ii) meso-tetrakis(4-N-methylpyridyl)porphyrin (PtTMPyP4) with polynucleotides [poly(dG-dC)]2 and [poly(dA-dT)]2 have been investigated using UV/visible, circular dichroism, and steady-state and time-resolved emission spectroscopy. PtTMPyP4 intercalates into [poly(dG-dC)]2 with K∼ 10(6) M(-1). When bound to [poly(dG-dC)]2 in aerated solution there is a six-fold emission enhancement with 18 nm red-shift in emission maximum. Emission lifetimes are biexponential. In the presence of [poly(dA-dT)]2 at least two distinct groove-binding modes are observed, depending on the binding ratio. In [poly(dA-dT)]2 the emission intensity increases by a maximum factor of 17 with no shift in the emission spectrum. Three exponentials were required for lifetime fitting. The lower extent of emission enhancement in the presence of [poly(dG-dC)]2 suggests that a slow electron transfer may take place to guanine, which is significantly less efficient than that previously observed for PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP). The results are compared to those previously recorded with free base H2TMPyP4.

Spectroscopic Studies on the Interaction of Co(II) Tetrapyridinoporphyrazine with Synthetic Polynucleotides and DNA

Interactions of cationic tetrakis (N,N´,N´´,N´´´-tetramethyltetra-3,4-pyridinoporphyrazinatocobalt(II) (Co(tmtppa)) with synthetic polynucleotides, poly(A-T), poly(G-C) and calf thymus DNA have been characterized in 5 mM phosphate buffer, pH 7.2, by optical absorption and fluorescence spectroscopy. The appearance of hypochromicity effect and the red shift in UV-Vis spectrum of porphyrazine was due to the interaction of both poly(A-T) and poly(G-C) which is similar to interaction of porphyrazine with DNA. The binding constants (K) were determined from the changes in the optical absorption spectra at various poly(G-C), poly(A-T) and DNA concentrations. According to the results, the values of K were 2.50 × 106 M-1, 2.25 × 106 M-1 and 2.25 × 105 M-1 for poly(A-T), poly(G-C) and DNA, respectively, at 25 oC. The thermodynamic parameters were calculated by the van’t Hoff equation. The positive values of the entropy and enthalpy suggest that coulombic interactions may play an important role in stabilization of the complex formed. The influence of the ionic strength was also investigated. It was concluded that the apparent binding constants decrease with increasing salt concentration. The fluorescence quenching of the DNA-ethidium bromide complex by the porphyrazine was also investigated. The values of the quenching constant (KSV) was determined by the Stern-Volmer equation. The results revealed groove binding mode of porphyrazine for both A-T and G-C rich region of polynucleotides of DNA.

Synthetic polynucleotides as endosomolytic agents and bioenergy sources

Nucleotides (NTs), such as adenosine triphosphate (ATP) and guanosine triphosphate (GTP), are signaling and bioenergy molecules to mediate a range of cellular pathways. We recently reported their significant endosomolytic activity. To evaluate whether polymeric NTs keep endosomolytic and bioenergetic functions of NTs in drug delivery and cell survival, NTs were polymerized by a coupling reaction to form polynucleotides (pNTs: pATP and pGTP) with their molecular weights around 500kDa. The cellular toxicity, indicated by IC50, of pNT was as low as that of corresponding monomeric NT. pNTs were degraded by an intracellular enzyme, alkaline phosphatase. Introduction of pNTs in a polycation-gene complex (polyplex) enhanced the extent of gene expression in cancerous, non-cancerous, and stem cells, up to 1500-fold higher than that of pNT-free polyplex. In addition, cells stored in a pATP solution resulted in a significantly higher survival rate (e.g., up to 20% increase) when exposed to low temperatures than pATP-free solution. The presence of pNT in polyplexes prevented the reduction of transfection efficiency induced by a low temperature. The findings in this study suggest that endosomolytic and bioenergetic pNTs serve as a non-toxic gene carrier component and protect cells from a cold shock or energy depletion.

Binding Geometry of Free Base and Mn(III)meso‐Tetrakis(N‐methylpyridium‐4‐yl)porphyrin to Various Duplex and Triplex DNAs

The binding mode of free base and Mn(III)meso‐tetrakis(N‐methylpyridium‐4‐yl)porphyrin (TMPyP and MnTMPyP) to various duplex and triplex synthetic polynucleotides was investigated by polarized light spectroscopy, specifically circular and linear dichroism (CD and LD) spectroscopy. Both TMPyP and MnTMPyP produced a similar positive CD spectrum in the Soret absorption region when bound to poly(dA)·poly(dT) duplex and poly(dA)·[poly(dT)]2 triplex. Considering that the third poly(dT) strand blocks the major groove, this result suggested that both porphyrins bound near the minor groove where the axial ligands of the central Mn(III) ion did not affect the binding mode. In contrast, TMPyP intercalated and MnTMPyP bound the exterior of poly(dG)·poly(dC) and poly(dI)·poly(dC) duplexes, as judged from the negative and positive CD signals, respectively. The CD signals for both intercalated TMPyP and externally bound MnTMPyP were greatly altered when bound to poly(dG)·poly(dC)·poly(dC)+ and poly(dI)·poly(dC)·poly(dC)+ triplexes, indicating that the binding modes of both porphyrins were affected by the presence of the third strand, poly(dC)+, which contrasted with the AT polynucleotides. This observation supported the conclusion that the external binding site of MnTMPyP at the GC and IC duplexes is the major groove. The reduced LD spectrum indicated that the angles of the two electric transition moments of both TMPyP and MnTMPyP in the Soret absorption region were 57° and 65° with respect to the local DNA helix axis when bound to the poly(dA)·[poly(dT)]2 triplex. On the other hand, a strong, wavelength‐dependent reduced LD was observed in the Soret region for both porphyrins when associated with CGC triplex, and the excessive tilt degree prevented any reasonable calculation of the angles.

Sequence specific binding of beta carboline alkaloid harmalol with deoxyribonucleotides: binding heterogeneity, conformational, thermodynamic and cytotoxic aspects.

BackgroundBase dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.Methodology/Principal FindingsBinding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.Conclusions/SignificanceStudies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC50 value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.