Analysis of mismatched DNA by mismatch binding ligand (MBL)–Sepharose affinity chromatography

Mismatch binding molecules (MBLs), strongly and selectively bound to the mismatched base pair in duplex DNA, were immobilized on Sepharose. Three MBL–Sepharose columns were prepared with three MBLs, naphthyridine dimer (ND), naphthyridine–azaquinolone (NA), and aminonaphthyridine dimer (amND), which exhibited different binding profiles to the mismatched base pairs. These three MBL–Sepharose columns showed characteristic elution profiles for DNA duplexes containing mismatched base pairs. The ND–Sepharose column separated the G–G and G–A mismatched DNA from fully matched duplexes. The NA–Sepharose column separated the A–A and G–A mismatched DNA from other DNA duplexes. The amND–Sepharose column separated the C–C mismatched DNA. These chromatographic profiles were very consistent with the binding preference of each MBL. By changing the elution conditions from sodium hydroxide to sodium chloride, MBL–Sepharose columns were also able to separate the mismatched DNA that weakly bound to the MBL from fully matched DNA duplex. Figure MBL-Sepharose affinity chromatography successfully separates the mismatched duplex DNA from fully matched duplex.     

DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

Marked dependence on carrier-ligand bulk but not on carrier-ligand chirality of the duplex versus single-strand forms of a DNA oligonucleotide with a series of G-Pt(II)-G intrastrand cross-links modeling cisplatin-DNA adducts

The N7-Pt-N7 adjacent G,G intrastrand DNA cross-link responsible for cisplatin anticancer activity is dynamic, promotes local "melting" in long DNA, and converts many oligomer duplexes to single strands. For 5'-d(A1T2G3G4G5T6A7C8C9C10A11T12)-3' (G3), treatment of the (G3)2 duplex with five pairs of [LPt(H2O)2]2+ enantiomers (L = an asymmetric diamine) formed mixtures of LPt-G3 products (1 Pt per strand) cross-linked at G3,G4 or at G4,G5 in all cases. L chirality exerted little influence. For primary diamines L with bulk on chelate ring carbons (e.g., 1,2-diaminocyclohexane), the duplex was converted completely into single strands (G3,G4 coils and G4,G5 hairpins), exactly mirroring results for cisplatin, which lacks bulk. In sharp contrast, for secondary diamines L with bulk on chelate ring nitrogens (e.g., 2,2'-bipiperidine, Bip), unexpectedly stable duplexes having two platinated strands (even a unique G3,G4/G4,G5 heteroduplex) were formed. After enzymatic digestion of BipPt-G3 duplexes, the conformation of the relatively nondynamic G,G units was shown to be head-to-head (HH) by HPLC/mass spectrometric characterization. Because the HH conformation dominates at the G,G lesion in duplex DNA and in the BipPt-G3 duplexes, the stabilization of the duplex form only when the L nitrogen adducts possess bulk suggests that H-bonding interactions of the Pt-NH groups with the flanking DNA lead to local melting and to destabilization of oligomer duplexes. The marked dependence of adduct properties on L bulk and the minimal dependence on L chirality underscore the need for future exploration of the roles of the L periphery in affecting anticancer activity.     

5'-Tethered stilbene derivatives as fidelity- and affinity-enhancing modulators of DNA duplex stability

A series of 5'-linked stilbene-DNA conjugates with different substituents in the distal aromatic ring of the stilbene was prepared, and the effect of the modifications on duplex stability was determined via UV-melting curves. A trimethoxystilbene derivative as a 5'-substituent increases duplex melting points by up to 12.2 degrees C per modification. With this alkoxystilbene substituent, terminal mismatches in DNA duplexes lower the melting point by up to 23.4 degrees C over the perfectly matched control, whereas terminal mismatches in unmodified DNA cause melting point depressions of no more than 6.1 degrees C. An aminomethylstilbene substituent linked to an oligopyrrolamide minor groove binder increases the melting point of an all-A/T decamer by up to 32.7 degrees C, thus shifting the melting point into a range typical for duplexes with statistical G/C-content. An affinity- and selectivity-enhancing effect was also observed when the trimethoxystilbene cap was employed on a small DNA microarray. The phosphoramidite of the trimethoxystilbene can be readily employed in automatic DNA synthesis, facilitating the generation of DNA chips with improved fidelity.     

Chemically induced hairpin formation in DNA monolayers

A naphthyridine dimer that binds specifically to G-G mismatches has been used to induce hairpin formation in oligonucleotides immobilized onto chemically modified gold surfaces. Surface plasmon resonance (SPR) imaging measurements of DNA microarrays were used to demonstrate that binding of the naphthyridine dimer to G-G mismatches within the stem portion of an immobilized 42-mer oligonucleotide could be used to induce hairpin formation that prevented hybridization of DNA complementary to the loop sequence. In addition, the selectivity of the naphthyridine dimer for G-G mismatches was verified through SPR imaging measurements of the hybridization adsorption of an 11-mer oligonucleotide to a four-component DNA array of zero- and single-base mismatch sequences.     

Investigation of matched and mismatched duplex DNA by electrospray ionization-mass spectrometry

In this research, the gas-phase stabilities of matched and mismatched duplex DNA were investigated by electrospray ionization-mass spectrometry (ESI-MS). The wild-type p53 duplex DNA [ds1, perfectly-matched (PM) DNA] was successfully distinguished from its three mutated DNAs [double-base mismatched DNA (DM)]. Moreover, the three DM DNAs were also well discriminated from each other using ESI-MS. Results show that the gas-phase thermodynamic stability of the DM DNAs decreased as the two mismatch spots moved closer. This implies that the dissociation of DM duplexes into two single strands prefers the mode "from middle to terminals".     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.     

Long-range cooperativity in molecular recognition of RNA by oligodeoxynucleotides with multiple C5-(1-propynyl) pyrimidines.

A heptamer composed of C5-(1-propynyl) pyrimidines (Y(p)'s) is a potent and specific antisense agent against the mRNA of SV40 large T antigen (Wagner, R. W.; Matteucci, M. D.; Grant, D.; Huang, T.; Froehler, B. C. Nat. Biotechnol. 1996, 14, 840-844). To characterize the role of the propynyl groups in molecular recognition, thermodynamic increments associated with substitutions in DNA:RNA duplexes, such as 5'-dCCUCCUU-3':3'-rGAGGAGGAAAU-5', have been measured by UV melting experiments. For nucleotides tested, an unpaired dangling end stabilizes unmodified and propynylated duplexes similarly, except that addition of a 5' unpaired rA is 1.4 kcal/mol more stabilizing on the propynylated, PODN:RNA, duplex than on the DNA:RNA duplex. Free energy increments for addition of single propynyl groups range from 0 to -4.0 kcal/mol, depending on the final number and locations of substitutions. A preliminary model for predicting the stabilities of Y(p)-containing hybrid duplexes is presented. Eliminating one amino group, and therefore a hydrogen bond, by substituting inosine (I) for guanosine (G), to give 5'-dC(p)C(p)U(p)C(p)C(p)U(p)U(p)-3':3'-rGAGIAGGAAAU-5', destabilizes the duplex by 3.9 kcal/mol, compared to 1.7 kcal/mol for the same change within the unpropynylated duplex. This 2.2 kcal/mol difference is eliminated by removing a single propynyl group three base pairs away. CD spectra suggest that single propynyl deletions within the PODN:RNA duplex have position-dependent effects on helix geometry. The results suggest long-range cooperativity between propynyl groups and provide insights for rationally programming oligonucleotides with enhanced binding and specificity. This can be exploited in developing technologies that are dependent upon nucleic acid-based molecular recognition.     

7,8-Dihydropyrido[2,3-d]pyrimidin-2-one; a bicyclic cytosine analogue capable of enhanced stabilisation of DNA duplexes

Incorporation of a bicyclic cytosine analogue, 3-beta-D-(2'-deoxyribofuranosyl)-7,8-dihydropyrido[2,3-d]pyrimidine, into synthetic DNA duplexes results in a greatly enhanced thermal stability (3-4 degrees C per modification) compared to the corresponding unmodified duplex.     

Insight into G[bond]T mismatch recognition using molecular dynamics with time-averaged restraints derived from NMR spectroscopy

Molecular dynamics (MD) simulations were conducted for a G[bond]T mismatch-containing DNA decamer, d(CCATGCGTGG)(2), and its Watson-Crick parent sequence, d(CCACGCGTGG)(2). Dynamics in unrestrained MD trajectories were in poor agreement with prior (13)C NMR studies. However, the accuracy of the trajectories was improved by the use of time-averaged interatomic distance restraints derived from (1)H NMR. Postprocess smoothing of the trajectories further improved accuracy. Comparison of restrained and smoothed trajectories of the two DNA molecules revealed distinct differences in dynamics. The major groove width of the mismatched oligomer was more variable over the course of the simulation compared to its parent sequence. Greater variability in helical parameters stretch and opening for the mismatches indicated less kinetically stable base pairing. Interbase helical parameters rise, roll, and tilt were also more variable in certain base steps involving mismatched bases. These dynamic differences between normal and G[bond]T mismatched DNA reflect differences in local flexibility that may play a role in mismatch recognition by the MutS. A potential alternate G[bond]T mismatch binding mode for MutS is also proposed.     

Computational study on the reaction mechanism of the key thermal [4 + 4] cycloaddition reaction in the biosynthesis of epoxytwinol A

[reaction: see text] The key [4 + 4] cycloaddition in the biosynthesis of epoxytwinol A has been established by theoretical calculations to comprise of three processes. The first step is formation of the C8-C8' bond generating a biradical intermediate. Next, rotation about the C8-C8' bond occurs, and finally the C1-C1' bond is formed. Biradicals stabilized by conjugation and two hydrogen bonds are essential for realization of this rare thermal [4 + 4] cycloaddition.     

Insertion of a bulky rhodium complex into a DNA cytosine-cytosine mismatch: an NMR solution study

The bulky octahedral complex Rh(bpy)2chrysi3+ (chrysi = 5,6-chrysenequinonediimine) binds single-base mismatches in a DNA duplex with micromolar binding affinities and high selectivity. Here we present an NMR solution study to characterize the binding mode of this bulky metal complex with its target CC mismatch in the oligonucleotide duplex (5'-CGGACTCCG-3')2. Both NOESY and COSY studies indicate that Rh(bpy)2chrysi3+ inserts deeply in the DNA at the mismatch site via the minor groove and with ejection of both destabilized cytosines into the opposite major groove. The insertion only minimally distorts the conformation of the oligonucleotide local to the binding site. Both flanking, well-matched base pairs remain tightly hydrogen-bonded to each other, and 2D DQF-COSY experiments indicate that all sugars maintain their original C2'-endo conformation. Remarkably, 31P NMR reveals that opening of the phosphate angles from a BI to a BII conformation is sufficient for insertion of the bulky metal complex. These results corroborate those obtained crystallographically and, importantly, provide structural evidence for this specific insertion mode in solution.     

Self-directed growth of benzonitrile line on H-terminated Si(001) surface

Using first-principles density-functional calculations we predict a self-directed growth of benzonitrile molecular line on a H-terminated Si(001) surface. The C[triple bond]N bond of benzonitrile reacts with a single Si dangling bond which can be generated by the removal of a H atom, forming one Si-N bond and one C radical. Subsequently, the produced C radical can be stabilized by abstracting a H atom from a neighboring Si dimer, creating another H-empty site. This H-abstraction process whose activation barrier is 0.65 eV sets off a chain reaction to grow one-dimensional benzonitrile line along the Si dimer row. Our calculated energy profile for formation of the benzonitrile line shows its relatively easier formation compared with previously reported styrene and vinylferrocene lines.     

Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs

We present the crystal structure of the DNA duplex formed by d(ATATATCT). The crystals contain seven stacked antiparallel duplexes in the asymmetric unit with A.T Hoogsteen base pairs. The terminal CT sequences bend over so that the thymines enter the minor groove and form a hydrogen bond with thymine 2 of the complementary strand in the Hoogsteen duplex. Cytosines occupy extra-helical positions; they contribute to the crystal lattice through various kinds of interactions, including a unique CAA triplet. The presence of thymine in the minor groove apparently contributes to the stability of the DNA duplex in the Hoogsteen conformation. These observations open the way toward finding under what conditions the Hoogsteen duplex may be stabilized in vivo. The present crystal structure also confirms the tendency of A.T-rich oligonucleotides to crystallize as long helical stacks of duplexes.     

Influence of intervening mismatches on long-range guanine oxidation in DNA duplexes

A systematic investigation of the efficiency of oxidative damage at guanine residues through long-range charge transport was carried out as a function of intervening base mismatches. A series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC approximately GG approximately GT approximately GA > AA > CC approximately TT approximately CA approximately CT. For all assemblies, damage found with the Delta-Ru diastereomer was found to be greater than with the Lambda-diastereomer. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from 1H NMR measurements of the imino proton exchange rates. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, damage ratios correlate most closely with base-pair lifetimes. Competitive hole trapping at the mismatch site does not appear to be a key factor governing the efficiency of transport through the mismatch. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.     

On the stability of double stranded nucleic acids

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.     

Towards the optimization of an optical DNA sensor: control of selectivity coefficients and relative surface affinities

Short single-stranded DNA (ssDNA) oligonucleotides can be grown on the surface of fused silica by automated nucleic acid synthesis. The immobilized ssDNA can be deposited at a desired average density. The density of ssDNA provides a controlled parameter that in combination with temperature, ionic strength and pH, can be used to define the selectivity of hybridization. Furthermore, the density of ssDNA can be used to control the affinity of complementary DNA so that it associates with the nucleic acids on the surface rather than areas that are not coated with ssDNA. The characteristic melt temperature observed for immobilized double-stranded DNA (dsDNA) 20mer shifts by up to 10 °C when a single base pair mismatch is present in the center of a target oligonucleotide. Optimization of quantitative analysis of such single base pair mismatches requires use of select experimental conditions to maximize the formation of the fully matched target duplex while minimizing the formation of the mismatched duplex. Results based on fiber optic biosensors that are used to study binding of fluorescein-labeled complementary DNA demonstrate that it is possible to achieve a selectivity coefficient of fully matched to single base pair mismatch of approximately 85-1, while maintaining >55% of the maximum possible signal that can be obtained from the fully matched target duplex.     

Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes

Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences and have investigated the properties of the duplexes formed from these strands upon binding of Cu(2+). Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu(2+), duplexes are formed even from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations demonstrate that at any temperature, one Cu(2+) ion binds a pair of Q-PNA strands that each contain one 8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate effect, which prevents the transformation in the presence of excess Cu(2+) of the 1:2 Cu(2+):Q-PNA complexes into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ(2)] moieties that are similar to the corresponding square planar synthetic complex formed between Cu(2+) and 8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a C-terminal l-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy. The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic acid nanostructures.