Sheared-type G(anti).C(syn) base-pair: a unique d(GXC) loop closure motif

Stable DNA loop structures closed by a novel G.C base-pair have been determined for the single-residue d(GXC) loops (X=A, T, G or C) in low-salt solution by high-resolution nuclear magnetic resonance (NMR) techniques. The closing G.C base-pair in these loops is not of the canonical Watson-Crick type, but adopts instead a unique sheared-type (trans Watson-Crick/sugar-edge) pairing, like those occurring in the sheared mismatched G.A or A.C base-pair, to draw the two opposite strands together. The cytidine residue in the closing base-pair is transformed into the rare syn domain to form two H-bonds with the guanine base and to prevent the steric clash between the G 2NH(2) and the C H-5 protons. Besides, the sugar pucker of the syn cytidine is still located in the regular C2'-endo domain, unlike the C3'-endo domain adopted for the pyrimidines of the out-of-alternation left-handed Z-DNA structure. The facile formation of the compact d(GXC) loops closed by a unique sheared-type G(anti).C(syn) base-pair demonstrates the great potential of the single-stranded d(GXC) triplet repeats to fold into stable hairpins.     

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.     

Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

Pulse sequences for detection of NH2···N hydrogen bonds in sheared G⋅A mismatches via remote, non-exchangeable protons

The non-detectability of NH...N hydrogen bonds in nucleic acids due to exchange broadened imino/amino protons has recently been addressed via the use of non-exchangeable protons for detecting internucleotide 2hJ(NN) couplings. In these applications, the appropriate non-exchangeable proton is separated by two bonds from the NH...N bond. In this paper, we extend the scope of this approach to protons which are separated by four bonds from the NH...N moiety. Specifically, we consider the case of the commonly occurring sheared G x A mismatch alignment, in which we use the adenine H2 proton to report on the (A)N6H6(1.2)...N3(G) hydrogen bond, in the presence of undetectable, exchange broadened N6H6(1.2) protons. Two sequences, the 'straight-through' (H6)N6N3H2 and 'out-and-back' H2N6N3 experiments, are presented for observing these correlations in H2O and D2O solution, respectively. The sequences are demonstrated on two uniformly 15N,13C labelled DNA samples: d(G1G2G3T4T5C6A7G8G9)2, containing a G3 x (C6-A7) triad involving a sheared G3 x A7 mismatch, and d(G1G2G3C4A5G6G7T8)4, containing an A5 x (G3 x G6 x G3 x G6) x A5 hexad involving a sheared G3 x A5 mismatch.     

Structural features and thermodynamics of the J4/5 loop from the Candida albicans and Candida dubliniensis group I introns

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5'GAAGG3'/3'UAAUU5', potentially contains two A.A pairs flanked by one G.U pair on one side and two G.U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A.A pairs with a cross-strand stack and only the G.U pair not adjacent to an A.A pair forming a static wobble G.U pair. The two G.U pairs adjacent to the tandem A.A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH(3))(6)(3+) stabilizes the loop by several kilocalories per mole at 37 degrees C, addition of Mg(2+) or Co(NH(3))(6)(3+) has no effect on the structure of the loop. The tandem G.U pairs provide a pocket of negative charge for Co(NH(3))(6)(3+) to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G.U pairs adjacent to internal loops.     

Molecular mechanics calculations on a triple stranded DNA involving C+.G-T and T.A+-C mismatched base triples

We have carried out molecular modeling of a triple stranded pyrimidine(Y). purine(R): pyrimidine(Y) (where ':' refers to Watson-Crick and '.' to Hoogsteen bonding) DNA, formed by a homopurine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC). Molecular mechanics calculations using NMR constraints have provided a detailed three dimensional structure of the triplex. The entire stretches of purine and the pyrimidine nucleotides have a conformation close to B-DNA. The three strands are held by the canonical C+.G:C and T.A:T hydrogen bonds. The structure also contains two mismatch C+.G-T and T.A+-C base triples which have been characterized for the first time. In the A+-C base-pair of the T.A+-C triple, both hydrogen donors are situated on the purine (A+(1N) and A+(6N)). We observe a unique hydrogen bonding interaction scheme in case of C+.G-T where one acceptor, G(60), is bonded to three donors (C+(3NH), C+(4NH2) and T(3NH)). Though the C+.G-T base triple is less stable than C+.G:C, it is significantly more stable than T.A:T. On the other hand, T.A+-C is as stable as the T.A:T base triad.     

A DNA hairpin with a single residue loop closed by a strongly distorted Watson-Crick G x C base-pair

Our previous NMR and modeling studies have shown that the single-stranded 19mer oligonucleotides d(AGCTTATC-ATC-GATAA GCT) -ATC- and d(AGCTTATC-GAT-GATAAGCT) -GAT- encompassing the strongest topoisomerase II cleavage site in pBR322 DNA could form stable hairpin structures. A new sheared base-pair, the pyrimidine-purine C x A, was found to close the single base -ATC- loop, while -GAT- displayed a flexible loop of three/five residues with no stabilizing interactions. Now we report a structural study on -GAC-, an analog of -GAT-, derived through the substitution of the loop residue T by C. The results obtained from NMR, non-denaturing PAGE, UV-melting, circular dichroism experiments and restrained molecular dynamics indicate that -GAC- adopts a hairpin structure folded through a single residue loop. In the -GAC- hairpin the direction of the G9 sugar is reversed relative to the C8 sugar, thus pushing the backbone of the loop into the major groove. The G9 x C11 base-pair closing the loop is thus neither a sheared base-pair nor a regular Watson-Crick one. Although G9 and C11 are paired through hydrogen bonds of Watson-Crick type, the base-pair is not planar but rather adopts a wedge-shaped geometry with the two bases stacked on top of each other in the minor groove. The distortion decreases the sugar C1'-C1' distance between the paired G9 and C11, to 8 A versus 11 A in the standard B-DNA. The A10 residue at the center of the loop interacts with the G9 x C11 base-pair, and seems to contribute to the extra thermal stability displayed by -GAC- compared to -GAT-. Test calculations allowed us to identify the experimental NOEs critical for inducing the distorted G.C Watson-Crick base-pair. The preference of -GAC- for a hairpin structure rather than a duplex is confirmed by the diffusion constant values obtained from pulse-field gradient NMR experiments. All together, the results illustrate the high degree of plasticity of single-stranded DNAs which can accommodate a variety of turn-loops to fold up on themselves.     

Unusual duplex formation in purine rich oligodeoxyribonucleotides

The purine rich oligodeoxyribonucleotides 1C, d(ATGACGGAATA) and 2C, d(ATGAGCGAATA) alone exhibit highly cooperative melting transitions. Analysis of the concentration dependence of melting, and electrophoretic studies indicate that these oligomers can form an unusual purine rich offset double helix. The unusual duplex is predicted to contain four A.T, two G.C, and four G.A mismatch base pairs as well as a single A base stacked on the 3' end of each chain of the helix. Other possible models for the duplex are unlikely because they are predicted to contain many base pairs of low stability. Changing the central sequence to CGG or GGG should destabilize the duplex and this is observed. The unusual duplex of 2C is more stable than the duplex of 1C indicating that the stability of G.A base pairs is quite sensitive to the surrounding sequence. Addition of 1C and 2C to their complementary pyrimidine strands results in normal duplexes of similar stability. We feel that the unusual duplexes are significantly stabilized by the intrinsic stacking tendency of purine bases.     

The conformational variability of an adenosine.inosine base-pair in a synthetic DNA dodecamer.

A crystal structure analysis of the synthetic deoxydodecamer d(CGCAAATTIGCG) which contains two adenosine.inosine (A.I) mispairs has revealed that, in this sequence, the A.I base-pairs adopt a A(anti).I(syn) configuration. The refinement converged at R = 0.158 for 2004 reflections with F greater than or equal to 2 sigma(F) in the range 7.0-2.5A for a model consisting of the DNA duplex and 71 water molecules. A notable feature of the structure is the presence of an almost complete spine of hydration spanning the minor groove of the whole of the (AAATTI)2 core region of the duplex. pH-dependent ultraviolet melting studies have suggested that the base-pair observed in the crystal structure is, in fact, a protonated AH+ (anti).I(syn) species and that the A.I base-pairs in the sequence studied display the same conformational variability as A.G mispairs in the sequence d(CGCAAATTGGCG). The AH+(anti).I(syn) base-pair predominates below pH 6.5 and an A(anti).I(anti) mispair is the major species present between pH 6.5 and 8.0. The protonated base-pairs are held together by two hydrogen bonds one between N6(A) and O6(I) and the other between N1(A) and N7(I). This second hydrogen bond is a direct result of the protonation of the N1 of adenosine. The ultraviolet melting studies indicate that the A(anti).I(anti) base-pair is more stable than the A(anti).G(anti) base-pair but that the AH+(anti).I(syn) base pair is less stable than its AH+(anti).G(syn) analogue. Possible reasons for this observation are discussed.     

6-Oxocytidine a novel protonated C-base analogue for stable triple helix formation.

2'-O-Methyl-3'-O-phosphoramidite building blocks of 6-oxocytidine 6 and its 5-methyl derivative 7, respectively, were synthesized and incorporated via phosphoramidite chemistry in 15 mer oligodeoxynucleotides [d(T72T7), S2; d(T73T7), S3] to obtain potential Py.Pu.Py triplex forming homopyrimidine strands. UV thermal denaturation studies and CD spectroscopy of 1:1 mixtures of these oligomers and a 21 mer target duplex [d(C3A7GA7C3)-d(G3T7CT7G3), D1] with a complementary purine tract showed a nearly pH-independent (6.0-8.0) triple helix formation with melting temperatures of 21-19 degrees C and 18.5-17.5 degrees C, respectively (buffer system: 50 mM sodium cacodylate, 100 mM NaCl, 20 mM MgCl2). In contrast, with the corresponding 15mer deoxy-C-containing oligonucleotide [d(T(7)1T7), S1] triplex formation was observed only below pH 6.6. Specificity for the recognition of Watson-Crick GC-base pairs was observed by pairing the modified C-bases of the 15mers with all other possible Watson-Crick-base compositions in the target duplex [d(C3A7XA7C3)-d(G3T7YT7G3), X = A,C,T; Y = T,G,A, D2-4]. Additionally, the Watson-Crick-pairing of the modified oligomers S2 and S3 was studied.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

Proton exchange and internal motions in two chromomycin dimer-DNA oligomer complexes.

Previous structural studies on the complexes of the chromomycin (CHR) dimer with duplexes of d(A1-A2-G3-G4-C5-C6-T7-T8) and of d(A1-G2-G3-A4-T5-C6-C7-T8) in solution [one Mg(II) and two drugs per duplex] are extended to hydrogen exchange measurements. Exchange of the OH8 proton of chromomycin, measured by real time proton-deuterium exchange, is very slow and requires dissociation of the complex, whose lifetime is thus determined. The lifetimes and apparent dissociation constants of base pairs are deduced from the catalysis of imino proton exchange by ammonia. The four central base pairs, which interact with the CHR chromophores in the minor groove (Gao & Patel, 1990), may open within the complex, but the opening rate is less than in the free duplex by one to two orders of magnitude. The activation energy for base-pair opening and the differences between the lifetimes of adjacent pairs suggest that single base-pair opening is the predominant imino proton exchange pathway in all cases. In the symmetrical complex of chromomycin with the first duplex, the lifetimes of the central base pairs (G3.C6 and G4.C5) are in the same range (52 and 29 ms, respectively, at 38 degrees C). In the asymmetrical complex formed with the second duplex, the base-pair lifetimes in the G2-G3-A4-T5 segment that interacts with the chromophore moiety are strongly increased. That of G3.C6 is particularly long. Above 50 degrees C, exchange of the G3 imino proton is opening limited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, HPO4(2-) or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2.G23 base pair was the most stable base pair in all the duplexes. The conserved U-1*G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2'-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1*G22, C-2*G23 and A2*U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed.     

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A-tracts

We have investigated by NMR the effects of NH(4)(+) on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B-DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](2), the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH(4)(+) binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH(4)(+) concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH(4)(+) concentration is kept larger than 1 M, the A.T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH(3) proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (W?rml?nder, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).     

Observation of water molecules within the bimolecular d(G₃CT₄G₃C)₂G-quadruplex

G-Rich oligonucleotides with cytosine residues in their sequences can form G-quadruplexes where G-quartets are flanked by G·C Watson-Crick base pairs. In an attempt to probe the role of cations in stabilization of a structural element with two G·C base pairs stacked on a G-quartet, we utilized solution state nuclear magnetic resonance to study the folding of the d(G(3)CT(4)G(3)C) oligonucleotide into a G-quadruplex upon addition of (15)NH(4)(+) ions. Its bimolecular structure exhibits antiparallel strands with edge-type loops. Two G-quartets in the core of the structure are flanked by a couple of Watson-Crick G·C base pairs in a sheared arrangement. The topology is equivalent to the solution state structure of the same oligonucleotide in the presence of Na(+) and K(+) ions [Kettani, A., et al. (1998) J. Mol. Biol.282, 619, and Bouaziz, S., et al. (1998) J. Mol. Biol.282, 637). A single ammonium ion binding site was identified between adjacent G-quartets, but three sites were expected. The remaining potential cation binding sites between G-quartets and G·C base pairs are occupied by water molecules. This is the first observation of long-lived water molecules within a G-quadruplex structure. The flanking G·C base pairs adopt a coplanar arrangement and apparently do not require cations to neutralize unfavorable electrostatic interactions among proximal carbonyl groups. A relatively fast movement of ammonium ions from the inner binding site to bulk with the rate constants of 21 s(-1) was attributed to the lack of hydrogen bonds between adjacent G·C base pairs and the flexibility of the T(4) loops.     

1H-1H correlations across N–H•••N hydrogen bonds in nucleic acids

In 2HJ(NN)-COSY experiments, which correlate protons with donor/acceptor nitrogens across Nd...HNa bonds, the receptor nitrogen needs to be assigned in order to unambiguously identify the hydrogen bond. For many situations this is a non-trivial task which is further complicated by poor dispersion of (Na,Nd) resonances. To address these problems, we present pulse sequences to obtain direct, internucleotide correlations between protons in uniformly 13C/15N labeled nucleic acids containing Nd...HNa hydrogen bonds. Specifically, the pulse sequence H2(N1N3)H3 correlates H2(A,omega1):H3(U,omega2) protons across Watson-CrickA-U and mismatched G.A base pairs, the sequences H5(N3N1)H1/H6(N3N1)H1 correlate H5(C,omega1)/H6(C,omega1):H1(G,omega2) protons across Watson-Crick G-C base pairs, and the H2(N2N7)H8 sequence correlates NH2(G,A,C;omega1):H8(G,A;omega2) protons across G.G, A.A, sheared G.A and other mismatch pairs. These 1H-1H connectivities circumvent the need for independent assignment of the donor/acceptor nitrogen and related degeneracy issues associated with poorly dispersed nitrogen resonances. The methodology is demonstrated on uniformly 13C/15N labeled samples of (a) an RNA regulatory element involving the HIV-1 TAR RNA fragment, (b) a multi-stranded DNA architecture involving a G.(C-A) triad-containing G-quadruplex and (c) a peptide-RNA complex involving an evolved peptide bound to the HIV-1 Rev response element (RRE) RNA fragment.     

Cyanine dyes as intercalating agents: kinetic and thermodynamic studies on the DNA/Cyan40 and DNA/CCyan2 systems

The interaction of cyanines with nucleic acids is accompanied by intense changes of their optical properties. Consequently these molecules find numerous applications in biology and medicine. Since no detailed information on the binding mechanism of DNA/cyanine systems is available, a T-jump investigation of the kinetics and equilibria of binding of the cyanines Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with CT-DNA is performed at 25 degrees C, pH 7 and various ionic strengths. Bathochromic shifts of the dye absorption band upon DNA addition, polymer melting point displacement (DeltaT = 8-10 degrees C), site size determination (n = 2), and stepwise kinetics concur in suggesting that the investigated cyanines bind to CT-DNA primary by intercalation. Measurements with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) reveal fair selectivity of CCyan2 toward G-C basepairs. T-jump experiments show two kinetic effects for both systems. The binding process is discussed in terms of the sequence D + S left arrow over right arrow D,S left arrow over right arrow DS(I) left arrow over right arrow DS(II), which leads first to fast formation of an external complex D,S and then to a partially intercalated complex DS(I) which, in turn, converts to DS(II), a more stable intercalate. Absorption spectra reveal that both dyes tend to self-aggregate; the kinetics of CCyan2 self-aggregation is studied by T-jump relaxation and the results are interpreted in terms of dimer formation.