Oligomeric Duplexes Research Articles

Detection of positional mismatch in oligonucleotide by electrochemical method

Detection of single base mismatch in DNA has been achieved by various techniques. However, the detection of position of that mismatch in the DNA duplex sequence has not been adequately addressed. In the present work, sensing of positional change of DNA base mismatch is achieved, with enhanced detection limit, using methylene blue as a redox indicator in electrochemical differential pulse voltammetric method (DPV). Interfacially synthesized Gold Polyaniline nanocomposites (Au-PAni) is coated on Pt electrode and attached with thiol modified single stranded DNA to construct the sensor electrode. Position sensitive detection of a single base mismatch in oligomeric duplexes is achieved here, with a detection limit for concentration as low as 10−8M for mixed oligomeric sequences and 10−6M for dA-dT duplexes. In this case, the sensitivity of the electrode is 0.12μApM−1cm−2. Reusable sensor electrode coated with Au-PAni nanocomposites was employed without compromising the sensitivity of the system. This is the first step in developing a highly sensitive method for detecting the effect of varying position of single base mismatch in the DNA.

Right-handed double-helix ultrashort DNA yields chiral nematic phases with both right- and left-handed director twist

Concentrated solutions of duplex-forming DNA oligomers organize into various mesophases among which is the nematic (N(∗)), which exhibits a macroscopic chiral helical precession of molecular orientation because of the chirality of the DNA molecule. Using a quantitative analysis of the transmission spectra in polarized optical microscopy, we have determined the handedness and pitch of this chiral nematic helix for a large number of sequences ranging from 8 to 20 bases. The B-DNA molecule exhibits a right-handed molecular double-helix structure that, for long molecules, always yields N(∗) phases with left-handed pitch in the μm range. We report here that ultrashort oligomeric duplexes show an extremely diverse behavior, with both left- and right-handed N(∗) helices and pitches ranging from macroscopic down to 0.3μm. The behavior depends on the length and the sequence of the oligomers, and on the nature of the end-to-end interactions between helices. In particular, the N(∗) handedness strongly correlates with the oligomer length and concentration. Right-handed phases are found only for oligomers shorter than 14 base pairs, and for the sequences having the transition to the N(∗) phase at concentration larger than 620mg/mL. Our findings indicate that in short DNA, the intermolecular double-helical interactions switch the preferred liquid crystal handedness when the columns of stacked duplexes are forced at high concentrations to separations comparable to the DNA double-helix pitch, a regime still to be theoretically described.

Association of the Minor Groove Binding Drug Hoechst 33258 with d(CGCGAATTCGCG)2: Volumetric, Calorimetric, and Spectroscopic Characterizations

We employed ultrasonic velocimetry, high-precision densimetry, circular dichroism and fluorescence spectroscopy, and isothermal titration calorimetry to characterize the binding of Hoechst 33258 to the d(CGCGAATTCGCG)(2) oligomeric duplex at 25 degrees C. We used this experimental combination to determine the full thermodynamic profile for the binding of Hoechst 33258 to the DNA. Specifically, we report changes in binding free energy, enthalpy, entropy, volume, and adiabatic compressibility accompanying the binding. We interpret our volumetric data in terms of hydration and evaluate the number of waters of hydration that become released to or taken up from the bulk. Our calorimetric data reveal that the drug-DNA binding event studied in this work is entropy-driven and proceeds with an unfavorable change in enthalpy. The favorable binding entropy predominantly results from hydration changes. In contrast to a large and positive change in hydrational entropy, the binding-induced change in configurational entropy is insignificant. The latter observation is consistent with the "lock-and-key" mode of minor groove binding.

Energetic diversity of DNA minor-groove recognition by small molecules displayed through some model ligand-DNA systems.

Energetics of interactions occurring in the model ligand-DNA systems constituted from distamycin A (DST), netropsin (NET) and the oligomeric duplexes d(GCAAGTTGCGATATACG)d(CGTATATCGCAACTTGC)=D#1 and d(GCAAGTTGCGAAAAACG)d(CGTTTTTCGCAACTTGC)=D#2 was studied by spectropolarimetry, UV-absorption spectroscopy and isothermal titration calorimetry. Model analysis of the measured signals was applied to describe individual and competitive binding in terms of populations of various species in the solution. Our results reveal several unprecedented ligand-DNA binding features. DST binds to the neighboring 5'-AAGTT-3' and 5'-ATATA-3' sites of D#1 statistically in a 2:1 binding mode. By contrast, its association to D#2 appears to be a 2:1 binding event only at the DST/D#2 molar ratios between 0 and 2 while its further binding to D#2 may be considered as a step-by-step binding to the unoccupied 5'-AAAAA-3' sites resulting first in DST3D#2 and finally in DST4D#2 complex formation. Competition between DST and NET binding shows that for the most part DST displaces NET from its complexes with D#1 and D#2. In contrast to the obligatory 1:1 binding of DST to the ligand-free 5'-AAAAA-3' sites observed at DST/5'-AAAAA-3' <1 the displacement of NET bound to the 5'-AAAAA-3' sites by added DST occurs even at the smallest additions of DST in a 2:1 manner. NET can also displace DST molecules but only those bound monomerically to the 5'-AAAAA-3' sites of DST3D#2. Actually, only half of these molecules can be displaced due to the simultaneous rebinding of the displaced DST to the unreacted 5'-AAAAA-3' sites in DST3D#2. Binding of DST and NET to D#1 and D#2 is an enthalpy driven process accompanied by large unfavorable (DST), small (NET) or large favorable (NET binding to 5'-AAAAA-3') entropy contributions and negative deltaCP degrees that are reasonably close to deltaCP degrees predicted from the calculated changes in solvent-accessible surface areas that accompany complex formation. Although various modes of DST and NET binding within D#1 and D#2 are characterized by significant energetic differences they seem to be governed by the same driving forces; the hydrophobic transfer of ligand from the solution into the duplex binding site and the accompanying specific non-covalent ligand-DNA and ligand-ligand interactions occurring within the DNA minor groove.

Measurement of nucleobase pKa values in model mononucleotides shows RNA-RNA duplexes to be more stable than DNA-DNA duplexes.

To understand why the RNA-RNA duplexes in general has a higher thermodynamic stability over the corresponding DNA-DNA duplexes, we have measured the pK(a) values of both nucleoside 3',5'-bis-ethyl phosphates [Etp(d/rN)pEt] and nucleoside 3'-ethyl phosphates [(d/rN)pEt] (N = A, G, C, or T/U), modeling as donors and acceptors of base pairs in duplexes. While the 3',5'-bis-phosphates, Etp(d/rN)pEt, mimic the internucleotidic monomeric units of DNA and RNA, in which the stacking contribution is completely absent, the 3'-ethyl phosphates, (d/rN)pEt, mimic the nucleotide at the 5'-end. The pK(a) values of the nucleobase in each of these model nucleoside phosphates have been determined with low pK(a) error (sigma = +/-0.01 to 0.02) by (1)H NMR (at 500 MHz) with 20-33 different pH measurements for each compound. This study has led us to show the following: (1) All monomeric DNA nucleobases are more basic than the corresponding RNA nucleobases in their respective Etp(d/rN)pEt and (d/rN)pEt. (2) The pK(a) values of the monomeric nucleotide blocks as well as Delta pK(a) values between the donor and acceptor can be used to understand the relative base-pairing strength in the oligomeric duplexes in the RNA and DNA series. (3) The Delta G*(pKa) of the donor and acceptor of the base pair in duplexes enables a qualitative dissection of the relative strength of the base-pairing and stacking in the RNA-RNA over the DNA-DNA duplexes. (4) It is also found that the relative contribution of base-pairing strength and nucleobase stacking in RNA-RNA over DNA-DNA is mutually compensating as the % A-T/U content increases or decreases. This interdependency of stacking and hydrogen bonding can be potentially important in the molecular design of the base-pair mimics to expand the alphabet of the genetic code.

Recognition of nine base pairs in the minor groove of DNA by a tripyrrole peptide-Hoechst conjugate.

A tripyrrole peptide-Hoechst conjugate (FPH-1) has been designed which recognizes nine dA/dT base pair A/T rich dsDNA sequences at subnanomolar concentrations and complexes its targets at near diffusion controlled rates to form a fluorescent product. Spectrofluorometric titrations show the stoichiometry of the complex to be (FPH-1)(2):dsDNA. Spectrofluorometric titrations were also employed to determine the product of the equilibrium constant for complexation (K(1)K(2)) of dsDNA by two FPH-1 molecules for 35 different oligomeric duplexes. Single base pair mismatches in the FPH-1 binding site were found to cause significant decreases in K(1)K(2) of 18- to 2300-fold. Thermal denaturation experiments provided similar results. Arguments are presented which favor the structure of the (FPH-1)(2):dsDNA minor groove complex to involve the two FPH-1 molecules in a slightly staggered, side-by-side, and antiparallel arrangement such that the bis-benzimidazole moiety of one FPH-1 molecule lies adjacent to the tripyrrole moiety of the second FPH-1 molecule.

Circular dichroic and kinetic differentiation of DNA binding modes of distamycin.

DNA binding modes of distamycin (DST) were investigated via comparative binding studies with oligomeric duplexes of the form d(GCG-X-GCG).d(CGC-Y-CGC), where Y is complementary to X and X = 4- or 5-base binding site. It was found that 1:1 and 2:1 drug-duplex complexes exhibit distinctly different circular dichroic (CD) spectral characteristics and can, thus, serve as diagnostic tools for binding mode differentiation. CD intensity profiles at 265 or 275 nm as a function of drug to DNA ratios can reveal the extent of binding cooperativity for 2:1 complex formation (i.e., the relative binding affinities of 2:1 vs 1:1) at a 5-base-paired binding site. Comparison of these profiles leads to the following qualitative ranking for the binding cooperativity for the studied sites: AAGTT, ATATA >/= AAACT > AATAA, AAATA, AAAGT > AATAT > TAAAA >/= AAATT >/= AAAAA >/= ATAAA, AAAAT. The plausibility of this ordering is strengthened by its agreement with the ranking established by earlier NMR studies on some of the sequences. The significantly slower DST dissociation kinetics of the 2:1 complexes as compared to those of 1:1 made the kinetic measurements of SDS-induced dissociation by the stopped-flow technique possible. The results indicate that the AAGTT site exhibits the slowest DST dissociation rate, with a characteristic time of 35 s. The rates of dissociation in general correlate reasonably well with the cooperativity order found via equilibrium CD measurements (the higher the binding cooperativity, the slower the rate of dissociation). Base sequence specific binding of DST was also found for the 1:1 complex formation at the 4-base-paired sites, with AAAA, TTTT, ATTT, and AAAT sequences exhibiting the highest binding affinities.

Sequence effects on the relative thermodynamic stabilities of B-Z junction-forming DNA oligomeric duplexes

Circular dichroism (CD) and ultraviolet absorption techniques were employed in characterizing the sequence-dependent thermodynamic stabilities of B-Z junction-forming DNA duplexes. The Watson strand of the duplexes has the general sequence (5meC-G)4-NXYG-ACTG (where N = A or G and XY represents all permutations of pyrimidine bases). Duplexes were generated by mixing stoichiometric amounts of the complementary strands. Circular dichroism studies indicate that each duplex is fully right-handed at low salt (e.g., 115 mM Na+) but undergoes a salt-induced conformational transition to a structure that possesses both left- and right-handed conformations at high salt (4.5 M Na+), and hence a B-Z junction. Optical melting studies of the DNA duplexes at fixed DNA concentration with total Na+ concentration ranging from 15 mM to 5.0 M were determined. A nonlinear dependence of the melting temperature (Tm) on [Na+] was observed. Thermodynamic parameters at Na+ concentrations of 115 mM and 4.5 M with a wide range of DNA concentrations were determined from UV optical melting studies via construction of van't Hoff plots. A change of a single dinucleotide within these duplexes significantly affected the helix stabilities. The experimentally obtained free energies for the duplex to single-strand transitions were in close agreement with predicted values obtained from two different methods.

Differential effects of the incorporation of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) on the binding of the transcription factors, AP-1 and TFIID, to their cognate target DNA sequences.

The thymidine analog, 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU), is incorporated into DNA in cell culture and in vivo. To investigate the effect of incorporation of FIAU into DNA on the binding of transcription factors, oligonucleotide duplexes which bind specifically to activator protein 1 (AP-1) or to TFIID were synthesized and binding of these oligonucleotides to their respective proteins was studied using gel-shift analysis. When thymidine at position -3, -1, 1 or 7 (relative to the first thymidine of the core binding sequence) was replaced with FIAU, binding to AP-1 was approximately 82, 28, 86 and 51%, respectively, of the binding to the non-substituted oligonucleotide to AP-1. When thymidine at position 3 or 5 (each adjacent to the center of dyad symmetry) was replaced with FIAU, binding to AP-1 was abrogated. Oligonucleotides containing FIAU at positions -1, 3 or 5, were much less able to compete with radiolabeled wild-type oligonucleotides for binding to AP-1. In contrast, the presence of FIAU, depending on its location, resulted in the increased binding of TFIID to its consensus target DNA sequence. These results indicate that incorporation of FIAU into DNA may induce local conformational changes resulting in the altered ability of transcriptional factors to bind to their cognate DNA sequences. Additional studies demonstrated that the presence of FIAU at a position 5' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by MseI.

Is the strong actinomycin D binding of d(5'CGTCGACG3') the consequence of end-stacking?

It has been reported that ACTD binds strongly and cooperatively to a non-GC containing self-complementary octamer d(CGTCGACG) with a 2:1 drug to duplex ratio (Synder et al., 1989). If one views the classic intercalative preference of ACTD for the 5'GpC3' sequence to be the drug favoring the 3'-side of dG, the possibility exists that the drug molecules may in fact stack on the G.C base pairs at both ends of this oligomeric duplex. To investigate this possibility, d(CGTCGACG) and several related oligomers resulting from replacing the terminal base(s) or appending with dT and/or dA are used in a comparative study employing equilibrium titration, thermal denaturation, kinetic, and various spectral measurements. Absorbance titrations at 20 degrees C confirm the strong and highly cooperative nature of ACTD binding to this octamer. The stoichiometric association constants for the binding of the first and second drugs were found to be 1 x 10(5) and 3.2 x 10(7) M-1, respectively. The base replacements of dG and dC at the respective ends resulted in a much weaker ACTD binding affinity, the loss of binding cooperativity, and much faster association and dissociation kinetics. These are consistent with the inability of the drug to stack on the 3'-side of dG due to base replacements. Appending the end(s) with dA and/or dT resulted in some diminution of binding affinity and cooperativity, appearance of slower association kinetic components, and unusually strong 7-amino-ACTD fluorescence enhancement for oligomers with dA or dT attached to dG at the 3'-terminal. To further support our postulate, studies were also made with d(CGACGTCG), which is related to the parent octamer by inverting the A.T pairs. It was found that, despite the altered internal sequence, this oligomer exhibits cooperative ACTD binding and kinetic characteristics very similar to those of the parent octamer, consistent with its ability to end-stack on the 3'-side of dG.

Importance of coulombic end effects on cation accumulation near oligoelectrolyte B-DNA: a demonstration using 23Na NMR

The local cation concentration at the surface of oligomeric or polymeric B-DNA is expected, on the basis of MC simulations (Olmsted, M. C., C. F. Anderson, and M. T. Record, Jr. 1989. Proc. Natl. Acad. Sci. USA. 86:7766-7770), to decrease sharply as either end of the molecule is approached. In this paper we report 23Na NMR measurements indicating the importance of this "coulombic" end effect on the average extent of association of Na+ with oligomeric duplex DNA. In solutions containing either 20-bp synthetic DNA or 160-bp mononucleosomal calf thymus DNA at phosphate monomer concentrations [P] of 4-10 mM, measurements were made over the range of ratios 1 < or = [Na]/[LP] < or = 20, corresponding to Na+ concentrations of 4-200 nM. The longitudinal 23Na NMR relaxation rates measured in these NaDNA solutions, Robs, are interpreted as population-weighted averages of contributions from "bound" (RB) and "free" (RF) 23Na relaxation rates. The observed enhancements of Robs indicate that RB significantly exceeds RF, which is approximately equal to the 23Na relaxation rate in an aqueous solution containing only NaCl. Under salt-fre-tconditions ([Na]/[P] = 1), where the enhancement in Robs is maximal, we find that Robs--RF in the solution containing 160-bp DNA is approximately 1.8 times that observed for the 20-bp DNA. For the 160-bp oligomer (which theoretical calculations predict to be effectively polyion-like), we find that a plot of Robs v. [P]/[Na] is linear, as observed previously for sonicated (approximately 700 bp) DNA samples. For the 20-bp oligonucleotide this plot exhibits a marked departure from linearity that can be fitted to a quadratic function of [P]/[Na]. Monte Carlo simulations based on a simplified model are capable of reproducing the qualitative trends in the 23Na NMR measurements analyzed here. In particular, the dependences of Robs--RF on DNA charge magnitude of Z(320 vs. 38 phosphates) and (for the 20-bp oligomer) on [Na]/[P] are well correlated with the calculated average surface concentration of Na+. Thus, effects of sodium concentration on RB appear to be of secondary importance. We conclude that 23Na NMR relaxation measurements are a sensitive probe of the effects of oligomer charge on the extent of ion accumulation near B-DNA oligonucleotides, as a function of [Na] and [P].

Spectroscopic and thermodynamic characterization of the interaction of N7-guanyl thioether derivatives of d(TGCTG*CAAG) with potential complements.

The oligomer d(TGCTGCAAG) corresponds to a region of bacteriophage M13mp18 DNA where mutations have been found to be induced by S-(2-chloroethyl)glutathione (glutathione, GSH) [Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., & Guengerich, F. P. (1991) J. Biol. Chem. 267, 6672-6679]. This oligomer was prepared with the central G replaced by S-(2-N7-guanylethyl)-GSH or N-acetyl-S-(2-N7-guanylethyl)Cys methyl ester; these derivatives were purified by HPLC and by affinity chromatography in the latter case. UV mixing and CD spectroscopy studies showed no evidence for preferred pairing of the S-(2-N7-guanylethyl)GSH moiety to any base other than C. UV melting studies of duplexes were performed with complementary strands containing the normal C, as well as the three mismatches (T, A, and G), across from the adducted base. Thermal stabilities were reduced in all cases when G was replaced by either N7-guanyl adduct; the C-containing complement was still the most stable. The reduced stability of the duplex d(TGCTG*CAAG)/d(CTTGCAGCA), where S-(2-N7-guanylethyl)-GSH corresponds to G*, was characterized by an increase in delta G zero of 1.4-2.0 kcal mol-1 (in the range of 25-37 degrees C) relative to the unadducted duplex. van't Hoff analysis of concentration-dependent melting experiments indicated that the delta H zero of the duplex was actually more favorable when this adduct was introduced (delta delta H zero = 13 kcal mol-1), but the decreased thermal stability was due to the entropic component. Similar results were observed when G* was N-acetyl-S-(2-N7-guanylethyl)Cys methyl ester. Under the conditions used, the overall relative stabilities of the oligomeric duplexes containing various base pairs do not indicate that S-(2-N7-guanylethyl)GSH would contribute to a higher frequency of T misinsertion than G. The possibility that ionization at the guanine N1 position may be involved in mutagenesis by N7-guanyl adducts is considered.

Duplex structure formation between oligo(dA)'s and oligo(dT)'s generated by thymine-specific interaction with netropsin.

The formation of oligomeric duplex molecules in the presence of the antibiotic netropsin in the series p(dA)n-p(dT)n is demonstrated using low-temperature CD measurements. Addition of Netropsin to mixtures of oligomers generates the same type of CD spectra as observed for poly(dA)-poly(dT) and maintains the duplex structure at temperatures at which base pairing of free oligomers is thermodynamically unstable. The shortest chain length forming a netropsin complex by thymine-specific interaction with the oligopeptide is represented by p(dA)4-p(dt)4. Studies with sequence isomers show that adjacent thymine residues strongly favour the complex formation with the oligopeptide.