Duplex Poly Research Articles

Binding properties of ruthenium(II) polypyridyl complexes [Ru(bpy)2(L)]2+(L=7-F-dppz and 7OCH3–-dppz) with poly(A)-poly(U) under dilute and molecular crowding conditions

Two ruthenium(II) polypyridyl complexes, [Ru(bpy)2(7-F-dppz)]2+ (Ru1, bpy = 2,2′-bipyridine, 7-F-dppz = 7-fluorodipyrido[3,2-a:2′,3′-c]phenazine) and [Ru(bpy)2(7OCH3–-dppz)]2+ (Ru2, 7OCH3–-dppz = 7‑methoxy-dipyrido-[3,2-a,2′,3′-c]-phenazine), were synthesized. The binding properties of Ru1 and Ru2 to duplex RNA poly(A)-poly(U) have been explored through spectroscopic techniques and viscometry. Spectral titration and viscosity experiments show that the binding mode of Ru1 and Ru2 to double-stranded RNA(dsRNA) in dilute solution and molecular crowded environment are intercalation, and that the binding affinity of Ru1 for poly(A)-poly(U) is greater than that of Ru2. Thermal denaturation experiments showed that two complexes have good thermal stability for dsRNA in two environments. Fluorescence titrations showed that Ru1 and Ru2 could act as molecular "light switch" for duplex poly(A)-poly(U) in both environments. Surprisingly, the binding effect of ligands and poly(A)-poly(U) was significantly reduced under crowded condition, and the thermal stability of RNA duplex was less significant than that of dilute solution. The reason of this result is due to the water activity in molecular crowded environment, the increased viscosity of the medium leading to a decrease in the affinity of the ligand for the RNA duplex, and also the electronic effect of the ligand on the binding affinity of the poly(A)-poly(U). Results of the present work facilitate our understanding of the two environments as well as the interaction between ruthenium(II) polypyridyl complexes and dsRNA is influenced by the electronegativity of the substituents.

New insights into self-structure induction in poly (rA) by Quinacrine through non-classical intercalation: Spectroscopic and theoretical perspectives

Self-structure induction in a single stranded polyriboadenylic acid [poly (rA)] is an auspicious physiological phenomenon which switches off protein production in tumor cells. In the present study, the self-structure induction process in poly (rA) moiety was thoroughly investigated using various steady state and time resolved techniques. Optical melting pattern directly evidenced the formation of self-structured assembly in single stranded poly (rA) upon complexation with quinacrine. Further, UV-absorption spectroscopic studies revealed that quinacrine binds to poly (rA) in co-operative fashion and the indication of intercalative mode of binding first came out with the involvement of around two base pairs of poly (rA) in the complexation. Experimental observations established the unconventional or non-classical intercalation of quinacrine molecule inside self-structured duplex poly (rA) moiety. This complexation was accompanied with negative enthalpy change and positive entropy change; suggesting strong van der Waals and the H-bonding interactions as the major governing forces in the complexation. Moreover, ionic strength dependent binding study established that the non-polyelectrolytic forces were the dominating forces. Further, the photo physical behavior of QN was authenticated using time dependent density functional theory (TDDFT) where both the ground and excited states were exploited.

Binding properties of a molecular “light switch” ruthenium(II) polypyridyl complex toward double- and triple-helical forms of RNA

To further develop new luminescent probes for RNA, a new ruthenium(II) polypyridyl complex [Ru(dmb)2dppz-idzo]2+ (dmb = 4,4′-dimethyl-2,2′-bipyridine, dppz-idzo = dppz-imidazolone) has been synthesized and characterized in this study. Binding properties of [Ru(dmb)2dppz-idzo]2+ to RNA duplex poly(A) · poly(U) and triplex poly(U) · poly(A) ∗ poly(U) have been explored by spectroscopic techniques and viscometry experiments. The binding modes of [Ru(dmb)2dppz-idzo]2+ to RNA duplex and triplex are intercalation as revealed from spectral titrations and viscosity experiments, while the binding strength of this complex to duplex structure is significantly greater than that of triplex structure. Fluorescence titrations indicate that [Ru(dmb)2dppz-idzo]2+ can act as a molecular “light switch” for both duplex poly(A) · poly(U) and triplex poly(U) · poly(A) ∗ poly(U), while [Ru(dmb)2dppz-idzo]2+ is more sensitive to poly(A) · poly(U) compared to poly(U) · poly(A) ∗ poly(U) and poly(U). Therefore, this complex can distinguish between RNA duplex, triplex and poly(U), and can as luminescent probes for the three RNAs used in this study. In addition, thermal denaturation studies show that [Ru(dmb)2dppz-idzo]2+ is able to significantly increase the Stabilization of RNA duplex and triplex. The results obtained in this study may contribute to further understanding of the binding of Ru(II) complexes with different structural RNAs.

Interaction of ruthenium(Ⅱ) polypyridyl complexes [Ru(phen)2(L)]2+ (L = PIP, p-HPIP and m-HPIP) with RNA poly(A)•poly(U): Each complex unexpectedly exhibiting a destabilizing effect on RNA

To further explore the binding properties of Ru(Ⅱ) polypyridine complexes with RNA, three Ru(Ⅱ) complexes [Ru(phen)2(PIP)]2+ (Ru1), [Ru(phen)2(p-HPIP)]2+ (Ru2), and [Ru(phen)2(m- HPIP)]2+ (Ru3) have been synthesized and characterized in this work. The binding properties of three Ru(Ⅱ) complexes with RNA duplex poly(A)•poly(U) have been investigated by spectral and viscosity experiments. These studies all support that these three Ru(Ⅱ) complexes bind to poly RNA duplex poly(A)•poly(U) by intercalation, and Ru1 without substituents has a stronger binding affinity for poly(A)•poly(U). Interestingly, the thermal melting experiments show that these three Ru(Ⅱ) complexes all destabilize RNA duplex poly(A)•poly(U), and the destabilizing effect can be explained by the conformational changes of duplex structure induced by intercalating agents. To the best of our knowledge, this work report for the first time a small molecule capable of destabilizing an RNA duplex, which reflects that the substitution effect of intercalated ligands has an important influence on the affinity of Ru(Ⅱ) complexes to RNA duplex, and that not all Ru(Ⅱ) complexes show thermal stability effects on an RNA duplex.

Singapore grouper iridovirus infection counteracts poly I:C induced antiviral immune response in vitro

Groupers are important mariculture fish in South China and Southeast Asian countries. However, the increasing frequency of infectious disease outbreaks has caused great economic losses in the grouper industry. Among these pathogens, Singapore grouper iridovirus (SGIV) infection causes high mortality in larval and juvenile stages of grouper. However, the mechanism underlying the action of viral manipulation on cellular immune response still remained largely uncertain. Here, using RNA-seq technology, we investigated the regulatory roles of SGIV infection on synthetic RNA duplex poly I:C induced immune response in vitro. Using reporter gene assays, we found that SGIV infection decreased poly I:C induced interferon promoter activation. Transcriptomic analysis showed that the mRNA expression levels of 2238 genes were up-regulated, while 1247 genes were down-regulated in poly I:C transfected grouper spleen (GS) cells. Interestingly, SGIV infection decreased the expression of 1479 up-regulated genes and increased the expression of 297 down-regulated genes in poly I:C transfected cells. The differentially expressed genes (DEGs) down-regulated by SGIV were directly related to immune, inflammation and viral infection, and JUN, STAT1, NFKB1, MAPK14A, TGFB1 and MX were the 6 top hub genes in the down-regulated DEGs’ protein-protein interaction (PPI) network. Furthermore, quantitative real-time PCR (qPCR) analysis confirmed that the interferon signaling and inflammatory-related genes, including cGAS, STING, TBK1, MAVS, TNF, IRAK4 and NOD2 were up-regulated by poly I:C stimulation, but all significantly down-regulated after SGIV infection. Thus, we speculated that SGIV infection counteracted poly I:C induced antiviral immune response and this ability helped itself to escape host immune surveillance. Together, our data will contribute greatly to understanding the potential immune evasion mechanism of iridovirus infection in vitro.

Binding and stabilization effect of arene ruthenium(Ⅱ) polypyridyl complexes toward the triple-helical RNA poly(U)•poly(A)⁎poly(U)

To determine the binding properties of η6‑arene Ru(Ⅱ) complexes with the RNA triplex poly(U)•poly(A)⁎poly(U), two arene Ru(Ⅱ) complexes, [(ŋ6-C6H6)Ru(m-npip)Cl]+ (Ru1) and [(ŋ6-C6H6)Ru(m-fpip)Cl]+ (Ru2), have been synthesized and characterized in this study. The binding of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)⁎poly(U) have been investigated by viscosity measurement and various spectroscopic methods. Results demonstrate that the RNA-binding behaviors of Ru1 and Ru2 are intercalation mode. From the analyses of electronic absorption spectra and fluorescence spectra, we observed that the binding affinity for Ru2 is remarkably stronger than that for Ru1. Furthermore, thermal denaturation studies reflect that Ru1 and Ru2 selectively stabilize the RNA triplex, among them, Ru1 can stabilize the Hoogsteen base-paired strand poly(U) (the third strand), while it is little effect on the Watson-Crick base-paired duplex poly(U)•poly(A) (the template duplex). Contrary to Ru1, Ru2 can stabilize both the template duplex and the third-strand, and Ru2 is more effective for stabilization of the third-strand. To our knowledge, this work is the first example of arene metal complexes interacting with triple-helical nucleic acids. The obtained results suggest that subtle differences of substituent on the intercalation ligand may be the important factor affecting on the binding properties of the both arene Ru(Ⅱ) complexes with the RNA triplex, when Ru1 and Ru2 contain the same ancillary ligands.

A naked-eye colorimetric molecular “light switch” based on ruthenium(II) polypyridyl complex [Ru(phen)2ttbd]2+ as binder and stabilizer for RNA duplex and triplex

Binding of [Ru(phen)2ttbd]2+ (phen = 1,10-phenanthroline, ttbd = 4-(6-propenylpyrido-[3,2-a]- phenzain-10-yl-benzene-1,2-diamine) to the RNA triplex poly(U-A*U) (herein “-” and “*” refer to the Watson-Crick and Hoogsteen binding, respectively) and the duplex poly(A-U) have been investigated by spectral technology and viscosity method. Analysis of spectral titrations and viscosity experiments as well as melting measurements suggest that [Ru(phen)2ttbd]2+ binds to the studied RNA triplex and duplex through intercalation, while its binding constant toward the triplex is greater than the duplex. Luminescent titrations indicate that [Ru(phen)2ttbd]2+ can act as a molecular “light switch” for the two RNAs and the switch effect can be detected by the naked-eye. Moreover, the “light switch” can be repeatedly cycled off and on by adjusting the pH of the solution, whereas color change in the case of the triplex is more significant compared with the duplex. To our knowledge, [Ru(phen)2ttbd]2+ is the first small molecule capable of serving as a pH-controlled reversible visual molecular “light switch” for both the RNA triplex poly(U-A*U) and duplex poly(A-U). Thermal denaturation experiments suggest that [Ru(phen)2ttbd]2+ can obviously increase the triplex stabilization, while it stabilizing third-strand is more marked in comparison with the template duplex of the triplex, indicating this complex preferentially binds to third-strand. The obtained results may be useful for understanding the binding of Ru(II) polypyridyl complexes to RNAs.

Ruthenium(II) polypyridyl complex [Ru(phen)2dppz-idzo]2+ as a colorimetric molecular "light switch" and powerful stabilizer for the RNA triplex poly(U)·poly(A)*poly(U).

The interaction of [Ru(phen)2dppz-idzo]2+ (phen = 1,10-phenanthroline, dppz-idzo = dppz-imidazolone) with triplex RNA poly(U)·poly(A)*poly(U) was carried out by using spectroscopic and viscometric techniques in this work. Luminescent titrations suggest that [Ru(phen)2dppz-idzo]2+ shows better selectivity for poly(U)·poly(A)*poly(U) compared with poly(U)·poly(A) and poly(U), this complex exhibits a "light switch" effect with an emission enhancement factor of about 123 in the presence of poly(U)·poly(A)*poly(U). Significantly, this "light switch" behavior could even be observed by the naked eye under irradiation with UV light. To our knowledge, [Ru(bpy)2dppz-idzo]2+ is the first small molecule able to serve as a colorimetric molecular "light switch" for the triplex poly(U)·poly(A)*poly(U). Combined with the spectral and viscometric results as well as [Ru(phen)2dppz-idzo]2+ stabilizing the template duplex poly(U)·poly(A), we speculate that [Ru(phen)2dppz-idzo]2+ prefers to bind with the Hoogsteen base-paired strand (the third strand) of the triplex, thus the intercalating [Ru(phen)2dppz-idzo]2+ stabilizing the third strand is more marked in comparison with the Watson-Crick base-paired duplex of the triplex. The results obtained here may be useful for understanding the interaction of triplex RNA poly(U)·poly(A)*poly(U) with small molecule, particularly ruthenium(II) complexes.

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.

Effect of ancillary ligands on the interaction of ruthenium(II) complexes with the triplex RNA poly(U)·poly(A)*poly(U)

Two new Ru(II) complexes with 1,8-naphthalimide group, [Ru(phen)2(pnip)]2+ (Ru1; phen=1,10-phenanthroline, pnip=2-[N-(p-phenyl)-1,8-napthalimide]imidazo[4',5'-f][1,10]phenanthroline) and [Ru(bpy)2(pnip)]2+ (Ru2; bpy=2,2′-bipyridine), have been synthesized and characterized. The interactions of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)*poly(U) (where • denotes the Watson–Crick base pairing and * denotes the Hoogsteen base pairing) were studied by various biophysical. Electronic spectra established that the binding affinity for Ru1 was greater than that for Ru2. Fluorescence and viscosity studies gave convincing evidence for a true intercalative binding of both complexes with the RNA triplex. UV melting studies confirmed that the two complexes could stabilize the triplex, whereas the effects of the two complexes on the stability of the Hoogsteen base-paired strand ploy(U) and the Watson–Crick base-paired duplex poly(U)•poly(A) of the triplex were different. In the case of Ru1, the increase of the thermal stability of the Hoogsteen base-paired strand was stronger than that of the Watson–Crick base-paired duplex. However, an opposite effect was observed in the case of Ru2. Circular dichroic studies suggested that the RNA triplex undergoes a conformational transition in the presence of Ru1, whereas the helicity of the RNA triplex still remains A-type in the presence of Ru2. The main results obtained here further advance our knowledge on the interaction of RNA triple-stranded structures with metal complexes, particularly ruthenium(II) complexes.

Interaction of 9-O-(ω-amino) alkyl ether berberine analogs with poly(dT)·poly(dA)*poly(dT) triplex and poly(dA)·poly(dT) duplex: a comparative study

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices. The present study has focused on the interaction of five 9-O-(ω-amino) alkyl ether berberine analogs with the DNA triplex poly(dT)·poly(dA)*poly(dT) and the parent duplex poly(dA)·poly(dT) studied using various biophysical techniques. Scatchard analysis of the spectral data indicated that the analogs bind both to the duplex and triplex in a non-cooperative manner in contrast to the cooperative binding of berberine to the DNA triplex. Strong intercalative binding to the DNA triplex structure was revealed from ferrocyanide quenching, fluorescence polarization and viscosity results. Thermal melting studies demonstrated higher stabilization of the Hoogsteen base paired third strand of the DNA triplex compared to the Watson-Crick strand. Circular dichroism studies suggested a stronger perturbation of the DNA triplex conformation by the alkaloid analogs compared to the duplex. The binding was entropy-driven in each case and the entropy contribution to free energy increased as the length of the alkyl side chain increased. The analogs exhibited stronger binding affinity to the triple helical structure compared to the parent double helical structure.

New aspects of the interaction of the antibiotic coralyne with RNA: coralyne induces triple helix formation in poly(rA)•poly(rU)

The interaction of coralyne with poly(A)•poly(U), poly(A)•2poly(U), poly(A) and poly(A)•poly(A) is analysed using spectrophotometric, spectrofluorometric, circular dichroism (CD), viscometric, stopped-flow and temperature-jump techniques. It is shown for the first time that coralyne induces disproportionation of poly(A)•poly(U) to triplex poly(A)•2poly(U) and single-stranded poly(A) under suitable values of the [dye]/[polymer] ratio (CD/CP). Kinetic, CD and spectrofluorometric experiments reveal that this process requires that coralyne (D) binds to duplex. The resulting complex (AUD) reacts with free duplex giving triplex (UAUD) and free poly(A); moreover, ligand exchange between duplex and triplex occurs. A reaction mechanism is proposed and the reaction parameters are evaluated. For CD/CP> 0.8 poly(A)•poly(U) does not disproportionate at 25°C and dye intercalation into AU to give AUD is the only observed process. Melting experiments as well show that coralyne induces the duplex disproportionation. Effects of temperature, ionic strength and ethanol content are investigated. One concludes that triplex formation requires coralyne be only partially intercalated into AUD. Under suitable concentration conditions, this feature favours the interaction of free AU with AUD to give the AUDAU intermediate which evolves into triplex UAUD and single-stranded poly(A). Duplex poly(A)•poly(A) undergoes aggregation as well, but only at much higher polymer concentrations compared to poly(A)•poly(U).

Destabilization and stabilization of poly(A) · poly(U) structure by platinum(II) compounds

On the basis of molecular biophysics, a methodology for the analysis of intramolecular structural order of the polynucleotide duplex poly(A).poly(U) has been developed. It was shown that the combination of circular dichroism spectroscopy with differential scanning calorimetry is an optimal approach, which ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) that can stabilize the structure and increase the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral remedy.

Spectroscopic and calorimetric studies on the binding of the phototoxic and cytotoxic plant alkaloid sanguinarine with double helical poly(A)

The interaction of the phototoxic and cytotoxic DNA binding plant alkaloid sanguinarine with double stranded polyadenylic acid has been investigated from multifaceted spectroscopic and calorimetric techniques. The hypochromic and bathochromic shifts in the absorption spectrum, and large enhancement of the fluorescence intensity of sanguinarine indicated strong binding of the alkaloid to the duplex poly(A). The corresponding intrinsic binding constant for the complexation, obtained from visible absorbance and fluorescence spectroscopic methods, respectively, was found to be in the order of 10 4 M −1. The binding was further characterized by strong energy transfer from the adenine base pairs to the alkaloid, significant polarization of the fluorescence of the alkaloid, stabilization against thermal strand separation of the duplex, and also resulting in remarkable conformational changes in poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Isothermal titration calorimetric and differential scanning calorimetric studies revealed the binding of the alkaloid to be exothermic and enthalpy driven. All these results together with fluorescence quenching experiments advance good evidence for the intercalation of the alkaloid sanguinarine into the duplex poly(A).

Characterization of Sac10a, a hyperthermophile DNA-binding protein from Sulfolobus acidocaldarius.

Sac10a is a member of a group of basic DNA-binding proteins thought to be important in chromatin structure and regulation in the archaeon Sulfolobus. We describe here the isolation, gene identification, and biophysical characterization of native Sac10a. The protein exists as a 23.8 kDa homodimer at pH 7 and unfolds with a T degrees of 122 degrees C. Dissociation of the dimer into folded globular subunits is promoted by decreased pH and salt concentration. Thermal unfolding of the monomeric subunits occurred with two transitions, indicating two independent domains. The dimer demonstrated a high affinity for duplex poly(dAdT) with a K(D) of 5 x 10(-)(10) M and a site size of 17 bp (in 0.15 M KCl, pH 7), with only weak binding (K(D) > 5 x 10(-)(6) M) to poly(dA)-poly(dT), poly(dGdC), poly(dG)-poly(dC), and Escherichia coli DNA under similar conditions. Binding to poly(dAdT) resulted in distortions in the DNA duplex that were consistent with overwinding as indicated by inversion of the CD spectrum of the DNA. The monomeric subunits are predicted to adopt a winged helix DNA-binding motif which dimerizes through formation of a two-stranded coiled coil involving an extended C-terminal helix with more than four heptad repeats (about 45 A in length). This is the first example of the conserved archaeal transcription regulator domain COG3432 to be characterized. Sequences for homologous proteins containing both COG3432 and predicted coiled coil domains occur in the genomes of both crenarchaeota (Sulfolobus, Pyrobaculum, Aeropyrum) and euryarchaeota (Methanosarcina, Methanococcus, Archaeoglobus, Thermoplasma), with multiple genes in some species. Sac10a shows no sequence similarity to the other Sulfolobus chromatin proteins Sac7d, Sac8, Sso10b2, and Alba.

DNA cross-linking by dehydromonocrotaline lacks apparent base sequence preference.

Pyrrolizidine alkaloids (PAs) are ubiquitous plant toxins, many of which, upon oxidation by hepatic mixed-function oxidases, become reactive bifunctional pyrrolic electrophiles that form DNA-DNA and DNA-protein cross-links. The anti-mitotic, toxic, and carcinogenic action of PAs is thought to be caused, at least in part, by these cross-links. We wished to determine whether the activated PA pyrrole dehydromonocrotaline (DHMO) exhibits base sequence preferences when cross-linked to a set of model duplex poly A-T 14-mer oligonucleotides with varying internal and/or end 5'-d(CG), 5'-d(GC), 5'-d(TA), 5'-d(CGCG), or 5'-d(GCGC) sequences. DHMO-DNA cross-links were assessed by electrophoretic mobility shift assay (EMSA) of 32P endlabeled oligonucleotides and by HPLC analysis of cross-linked DNAs enzymatically digested to their constituent deoxynucleosides. The degree of DNA cross-links depended upon the concentration of the pyrrole, but not on the base sequence of the oligonucleotide target. Likewise, HPLC chromatograms of cross-linked and digested DNAs showed no discernible sequence preference for any nucleotide. Added glutathione, tyrosine, cysteine, and aspartic acid, but not phenylalanine, threonine, serine, lysine, or methionine competed with DNA as alternate nucleophiles for cross-linking by DHMO. From these data it appears that DHMO exhibits no strong base preference when forming cross-links with DNA, and that some cellular nucleophiles can inhibit DNA cross-link formation.

Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation.

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)*poly(dT) into triplex poly(dA)*poly(dT)*poly(dT) and a poly(dA) self-structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)16*(dT)16 by coralyne reverts over the course of hours if the sample is maintained at 4 degrees C. Coralyne-disproportioned (dA)32. (dT)(32), on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)16*(dT)16 sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)16 self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)n self-structure] binds coralyne in a length-dependent manner.

Sequence and structural selectivity of nucleic acid binding ligands

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).

Complete disproportionation of duplex poly(dT)middle dotpoly(dA) into triplex poly(dT)middle dotpoly(dA)middle dotpoly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)*poly(dA). That is, coralyne causes the strands of duplex poly(dT)*poly(dA) to repartition into equal molar equivalents of triplex poly(dT)*poly(dA)*poly(dT) and poly(dA). Poly(dT)*poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4 degrees C. However, disproportionation readily occurs upon heating above 35 degrees C and is not reversed by subsequent cooling. A titration of poly(dT)*poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47 degrees C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)*poly(dA)*poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A*A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)*poly(dA).

Sequence and structural selectivity of nucleic acid binding ligands.

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).