Improved selectivity for the binding of naphthyridine dimer to guanine-guanine mismatch.

Naphthyridine dimer composed of two 2-amino-1,8-naphthyridines and a connecting linker strongly binds to guanine-guanine (G-G) mismatch in duplex DNA. In order to improve G-G selectivity for the binding, we have examined structure modification of the linker. A new naphthyridine dimer possessing 3,6-diazaoctanedioic acid linker binds to G-G mismatch with an association constant of 1.18 x 10(7) M(-1), which is somewhat weaker than that of the original naphthyridine dimer having a shorter connecting linker. However, the binding of the modified naphthyridine dimer to G-A mismatch was almost negligible as compared to that of the original. This results in a net increase of the selectivity for the binding to G-G mismatch by 4-folds.     

The binding of guanine-guanine mismatched DNA to naphthyridine dimer immobilized sensor surfaces: kinetic aspects

Naphthyridine dimer composed of two naphthyridine chromophores and a linker connecting them strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. The kinetics for the binding of the G-G mismatch to the naphthyridine dimer was investigated by surface plasmon resonance assay. The sensor surface was prepared by immobilizing naphthyridine dimer through a long poly(ethylene oxide) linker with the ligand density of 9.1 x 10(-12) fmolnm(-2). The kinetic analyses revealed that the binding of the G-G mismatch was sequence dependent on the flanking base pairs, and the G-G mismatches flanking at least one G-C base pair bound to the surface via a two-step process with a 1:1 DNA-ligand stoichiometry. The first association rate constant for the binding of the G-G mismatch in the 5'-CGG-3'/3'-GGC-5' sequence to the naphthyridine dimer-immobilized sensor surface was 3.2 x 10(3)M(-1)s(-1) and the first dissociation rate constant was 1.4 x 10(-2)s(-1). The association and dissociation rate constants for the second step were insensitive to the flanking sequences, and were almost of the same order of magnitude as the first dissociation rate constant. This indicates that the second step had only a small energetic contribution to the binding. The association constant calculated from kinetic parameters was 2.7 x 10(5)M(-1), which is significantly smaller than the apparent association constants obtained from experiments in solution. Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry on the complex produced from the G-G mismatch and naphthyridine dimer showed the formation of the 1:1 complex and a 1:2 DNA-ligand complex in solution. The latter complex became the dominant complex when a six-fold excess of naphthyridine dimer was added to DNA.     

Highly sensitive detection of GG mismatched DNA by surfaces immobilized naphthyridine dimer through poly(ethylene oxide) linkers

Naphthyridine dimer is a unique molecule that strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. We have synthesized naphthyridine dimers possessing a different length of poly(ethylene oxide) (PEO) linker, and immobilized them to CM5 sensor chip to carry out a surface plasmon resonance (SPR) assay of DNA duplexes containing a single base mismatch. The sensitivity of the sensor remarkably increased with increasing numbers of PEO units incorporated into the linker. With the sensor surface immobilized naphthyridine dimer for 1.5 x 10(3) response unit (RU) through three PEO units, the distinct SPR signal was observed at a concentration of 1 nM of the 27-mer G-G mismatch.     

Scanning of guanine-guanine mismatches in DNA by synthetic ligands using surface plasmon resonance

Here we have designed and synthesized ligands that specifically bind with high affinity (K(d) = 53 nM) to the guanine (G)-guanine mismatch, one of four types of single-nucleotide polymorphism (SNP). Detection of the G-G mismatch was performed by a surface plasmon resonance (SPR) assay using a sensor chip carrying the G-G specific ligand on its surface. The accuracy of the G-G mismatch detection by the SPR sensor was demonstrated by a marked SPR response obtained only for the DNA containing the G-G mismatch. DNAs containing G-A and G-T mismatches, as well as a fully matched duplex, produced only a weak response. Furthermore, this assay was found applicable for the detection of SNP existing in PCR amplification products of a 652-nucleotide sequence of the HSP70-2 gene.     

A new ligand binding to G-G mismatch having improved thermal and alkaline stability

Naphthyridine dimer (ND) specially binds to guanine-guanine (G-G) mismatch in duplex DNA. In order to improve the thermal and alkaline stability and binding ability of the ligand, we have examined structural modification of the linker. A new ligand (NNC) possessing 2-amino-1,8-naphthyridines and a carbamate linker is much more thermally stable than ND. The half-life of NNC is 2.5 times longer than that of ND at 80 degrees C. NNC is also much more stable than ND under alkaline conditions. In addition, NNC binds to G-G mismatch more strongly than ND. The improved stability and the binding of NNC to the G-G mismatch would be suitable for the practical use of NNC-immobilized sensor.     

NMR study of a novel RNA quadruplex structure.

The structure of an RNA oligomer, r (GGAGGUUUUGGAGG) (R14-2) whose G-G steps are separated by adenine and uracil residues has been investigated by NMR. In the presence of 20 mM K+, a novel dimeric multiplex architecture is adopted by two strands of R14-2. In each strand a UUUU loop and two A residues connect four parallel G-G steps that pair-align into two tetrads. One of the tetrads is further pair-aligned by two A residues through the sheared mismatch and a novel hexad is subsequently formed. Two hexads coming from two different strands stack to make a dimeric multiplex. All of the guanosine and adenosine residues take an anti conformation.     

The thermodynamics of 3'-terminal pyrene and guanosine for the design of isoenergetic 2'-O-methyl-RNA-LNA chimeric oligonucleotide probes of RNA structure

To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.     

Dimer of 2,7-diamino-1,8-naphthyridine for the detection of mismatches formed by pyrimidine nucleotide bases

Discrimination of base mismatches from normal Watson-Crick base pairs in duplex DNA constitutes a key approach to the detection of single nucleotide polymorphisms (SNPs). We have developed a sensor for a surface plasmon resonance (SPR) assay system to detect G-G, A-A, and C-C mismatch duplexes by employing a surface upon which mismatch-binding ligands (MBLs) are immobilized. We synthesized a new MBL consisting of 2,7-diamino-1,8-naphthyridine (damND) and immobilized it onto a CM5 sensor chip to carry out the SPR assay of DNA duplexes containing a single-base mismatch. The SPR sensor with damND revealed strong responses to all C-C mismatches, and sequence-dependent C-T and T-T mismatches. Compared to ND- and naphthyridine-azaquinolone hybrid (NA)-immobilized sensor surfaces, with affinity to mismatches composed of purine nucleotide bases, the damND-immobilized surface was useful for the detection of the mismatches composed of pyrimidine nucleotide bases.     

Expanding chemical space of DNA-binding molecules with three base-binding units

A new molecule NC3-3 designed to expand chemical space of parent molecule NCD by adding the third base-binding unit was reported. NC3-3 bound to the G-G mismatch in the 5′-CGG-3′/5′-CGG-3′ motif but not to that in 5′-GGC-3′/5′-GGC-3′. This binding selectivity is similar to that reported for NCD. Fluorimetric screening of NCD and NC3-3 to dsDNA library containing yGw/xGz motifs showed that NC3-3 still kept the sequence selectivity as we observed for NCD-binding. The third naphthyridine heterocycle in NC3-3 affected the mode of the binding, but a little effect on the sequence selectivity.     

DNA mismatch binding activities in Chlorella pyrenoidosa extracts and affinity isolation of G-T mismatch binding proteins.

DNA mismatch recognition proteins contained in the extracts of unicellular alga Chlorella pyrenoidosa were isolated by affinity adsorption and 2-D gel electrophoresis. Incubation of the algal extracts with a 38-mer duplex oligonucleotide carrying a single DNA simple mispair generated a few gel retardation complexes. G-T mispair was recognized significantly better than C-T, G-G, G-A, and C-C mispairs by the algal extracts and these extracts bound very weakly to G-A and C-C mispairs, displaying a universal trend of mismatch binding efficiency. The levels of mismatch recognition complexes were slightly increased in the presence of 1 mM ATP. Two 13-kDa G-T binding polypeptides possessing pIs of 5.3 and 5.5 were isolated after resolving affinity-captured proteins by 2-D gel electrophoresis and the two factors were found to bind 5.5- and 2.8-fold stronger to heteroduplex than to homoduplex DNA, respectively. No proteins significantly homologous to the two algal G-T binding proteins were found by peptide mass fingerprinting (PMF). The sequence of a peptide generated from trypsin-cleavage of one G-T binding factor (pI 5.5) could be aligned with the amino acid sequences that form the C-terminal active sites of human and mouse mismatch-specific uracil/thymine-DNA glycosylases, suggesting the possibility of this factor as an algae- or a Chlorella-specific DNA mismatch glycosylase.     

DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence

Expansion of the triplet repeat DNA sequence d[CGG]n.d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C-C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C-C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C-C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4].d[N4N3N2N1Cm1m2m3m4] sequence, where M-m and N-n are complementary base pairs, the amount of crosslink increased with increasing G-C content of the eight base pairs neighboring the C-C mismatch and with the proximity of the G-C pairs to the C-C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C-C pair flanked by G-C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C-C pair flanked by A-T base pairs, the simulations suggested local opening of the duplex around the C-C pair, making it a less effective target for mechlorethamine.     

Klenow exo-, as opposed to exo+, traverses through G-G:C triplex by melting G-G base pairs

G-G base-paired hairpin DNA structures on template strands offer potential "road-blocks" to a traversing polymerase. Klenow polymerase (exo+) pauses while replicating through G-G base-paired hairpin DNA due to the generation of G-G:C triplex. However, exonuclease-deficient Klenow traverses through de novo generated G-G:C triplexes leading to full-length C:G duplexes. Alleviation of such road-blocks by exo- Klenow ensues faster at lower Mg2+, a kinetic effect consistent with the role of Mg2+ in stabilizing G-G:C triplex fold. The ability of exonuclease-deficient polymerase to go past the de novo generated G-G:C triplexes suggests that the "idling" of exo+ polymerase at G-G road-block is due to the reiterative polymerase/exonuclease action. The full-length replication product carrying a C(n)-G(n) duplex at one end is further "expanded" by exo- Klenow through C-strand "slippage" leading to the generation of C+-G:C triplex, which is exemplified by the premature arrest of the same at low pH that further stabilizes the C+-G:C triplex.     

Evaluation of mismatch-binding ligands as inhibitors for Rev-RRE interaction

Drugs targeting the stem-loop IIB of Rev responsible element (RRE) of HIV-1 mRNA are potential therapeutic agents for HIV-1 infection. The stem loop is characterized by an internal loop consist of consecutive G-G and G-A mismatches, which is the single binding site for Rev protein for nuclear export of viral mRNA. We report here that ligands binding to G-G and G-A mismatches in duplex DNA also bind to the internal loop in competition with Rev peptide and lead to the dissociation of pre-formed Rev-RRE complex in a model system.     

Molecular cloning of zebrafish (Danio rerio) MutS homolog 6(MSH6) and noncoordinate expression of MSH6 gene activity and G-T mismatch binding proteins in zebrafish larvae

Eukaryotic MutS homolog 6(MSH6) is a DNA mismatch recognition protein associated with mismatch repair of simple base-base mispairs and small insertion-deletion loops. As replication or recombination errors generated during embryonic development of living organisms should be efficiently corrected to maintain the integrity of genetic materials, we attempted to study MSH6 gene expression in developing zebrafish (Danio rerio) and the influence of MSH6 expression on the production of mismatch binding factors. A full-length cDNA encoding zebrafish MSH6 (zMSH6) was first obtained by rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of zMSH6 shares 57% and 56% identity with human and mouse MSH6, respectively. The 190-kDa recombinant zMSH6 containing 1,369 amino acids bound preferentially to a heteroduplex than to a homoduplex DNA. Northern blot and semiquantitative RT-PCR analysis detected apparent levels of zMSH6 mRNA expression in 12 and 36-hr-old zebrafish embryos, while this expression in 84-hr-old larvae was dramatically reduced to 23% of that in 12-hr-old embryos when beta-actin mRNA was constitutively synthesized. Incubation of G-T and G-G heteroduplex probes with 12 to 60-hr-old zebrafish extracts produced predominantly high-shifting binding complexes with very similar band intensity. Although low in zMSH6 mRNA production, the extracts of 84-hr-old larvae interacted significantly stronger than the embryonic extracts with both G-T and G-G mispairs, producing high and low-shifting complexes. Heteroduplex-recognition proteins in 108-hr-old larvae gave a similar pattern of mismatch binding. The intensities of G-T complexes produced by 84 and 108-hr-old zebrafish extracts were 2.5 to 3-fold higher than those of G-G complexes. Our data indicate that the production of efficient MSH6-independent binding factors, particularly G-T-specific recognition proteins, is upregulated in zebrafish at the larval stage when MSH6 gene activity is downregulated.     

Interaction of nick-directed DNA mismatch repair and loop repair in human cells

In human cells, large DNA loop heterologies are repaired through a nick-directed pathway independent of mismatch repair. However, a 3'-nick generated by bacteriophage fd gene II protein heterology is not capable of stimulating loop repair. To evaluate the possibility that a mismatch near a loop could induce both repair types in human cell extracts, we constructed and tested a set of DNA heteroduplexes, each of which contains a combination of mismatches and loops. We have demonstrated that a strand break generated by restriction endonucleases 3' to a large loop is capable of provoking and directing loop repair. The repair of 3'-heteroduplexes in human cell extracts is very similar to that of 5'-heteroduplex repair, being strand-specific and highly biased to the nicked strand. This observation suggests that the loop repair pathway possesses bidirectional repair capability similar to that of the bacterial loop repair system. We also found that a nick 5' to a coincident mismatch and loop can apparently stimulate the repair of both. In contrast, 3'-nick-directed repair of a G-G mismatch was reduced when in the vicinity of a loop (33 or 46 bp between two sites). Increasing the distance separating the G-G mismatch and loop by 325 bp restored the efficiency of repair to the level of a single base-base mismatch. This observation suggests interference between 3'-nick-directed large loop repair and conventional mismatch repair systems when a mispair is near a loop. We propose a model in which DNA repair systems avoid simultaneous repair at adjacent sites to avoid the creation of double-stranded DNA breaks.     

The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs

The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes. These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain. Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments. Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction. The rDNA is G-rich and has considerable potential for G-G pairing. Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase.     

A dimeric DNA interface stabilized by stacked A.(G.G.G.G).A hexads and coordinated monovalent cations

We report on the identification of an A.(G.G.G.G).A hexad pairing alignment which involves recognition of the exposed minor groove of opposing guanines within a G.G.G.G tetrad through sheared G.A mismatch formation. This unexpected hexad pairing alignment was identified for the d(G-G-A-G-G-A-G) sequence in 150 mM Na(+) (or K(+)) cation solution where four symmetry-related strands align into a novel dimeric motif. Each symmetric half of the dimeric "hexad" motif is composed of two strands and contains a stacked array of an A.(G.G.G.G).A hexad, a G.G.G.G tetrad, and an A.A mismatch. Each strand in the hexad motif contains two successive turns, that together define an S-shaped double chain reversal fold, which connects the two G-G steps aligned parallel to each other along adjacent edges of the quadruplex. Our studies also establish a novel structural transition for the d(G-G-A-G-G-A-N) sequence, N=T and G, from an "arrowhead" motif stabilized through cross-strand stacking and mismatch formation in 10 mM Na(+) solution (reported previously), to a dimeric hexad motif stabilized by extensive inter-subunit stacking of symmetry-related A.(G.G.G.G).A hexads in 150 mM Na(+) solution. Potential monovalent cation binding sites within the arrowhead and hexad motifs have been probed by a combination of Brownian dynamics and unconstrained molecular dynamics calculations. We could not identify stable monovalent cation-binding sites in the low salt arrowhead motif. By contrast, five electronegative pockets were identified in the moderate salt dimeric hexad motif. Three of these are involved in cation binding sites sandwiched between G.G.G. G tetrad planes and two others, are involved in water-mediated cation binding sites spanning the unoccupied grooves associated with the adjacent stacked A.(G.G.G.G).A hexads. Our demonstration of A.(G. G.G.G).A hexad formation opens opportunities for the design of adenine-rich G-quadruplex-interacting oligomers that could potentially target base edges of stacked G.G.G.G tetrads. Such an approach could complement current efforts to design groove-binding and intercalating ligands that target G-quadruplexes in attempts designed to block the activity of the enzyme telomerase.     

A dimeric RNA quadruplex architecture comprised of two G:G(:A):G:G(:A) hexads,G:G:G:G tetrads and UUUU loops

Using CD and NMR, we determined the structure of an RNA oligomer, r(GGAGGUUUUGGAGG) (R14), comprising two GGAGG segments joined by a UUUU segment. A modified quadruplex structure was observed for r(GGAGGUUUUGGAGG) in solution even in the absence of K(+). An unusually stable dimeric RNA quadruplex architecture formed from two strands of r(GGAGGUUUUGGAGG) at low K(+) concentration is reported here. In each strand of r(GGAGGUUUUGGAGG), two sets of successive turns in the GGAGG segments and turns at both ends of the UUUU loops drive four G-G steps to align in a parallel manner, a core with two stacked G-tetrads being formed. Two adenine bases bind to two edges of one G:G:G:G tetrad through the sheared G:A mismatch augmenting the tetrad into a G:G(:A):G:G(:A) hexad. Thus, one molecule of r(GGAGGUUUUGGAGG) folds into a modified quadruplex comprising a G:G:G:G tetrad, a UUUU double-chain reversal loop and a G:G(:A):G:G(:A) hexad. Two such molecules further associate by stacking through the dimeric hexad-hexad interface with a rotational symmetry. The ribose rings of most nucleotides take S (close to C2'-endo) puckering, which is unusual for an RNA. K(+) can increase the stability of this quadruplex structure; the number of bound K(+) was estimated from the results of the titration experiment. Besides G:G and G:A mismatches, a network of hydrogen bonds including O4'-NH(2) and C-H..O hydrogen bonds, and the extensive base stacking contribute to the high thermodynamic stability of R14. Our results could provide the stereochemical and thermodynamic basis for elucidating the biological role of the GGAGG-containing RNA segments abundantly existing in various RNAs. Relevance to quadruplex-mediated mRNA-FMRP binding and HIV-1 genome RNA dimerization is discussed.     

Temperature-gradient DNA probe chromatography of nucleic acids on nonporous supports.

Temperature-gradient DNA probe chromatography of nucleic acids on a nonporous support with a homogeneous particle size of 2.5 microns showed a higher base sequence discriminating power and a larger linear capacity than that on a porous support with a larger and less homogeneous particle size. The resolution on the nonporous support was high enough to separate samples with a single-base mismatch of the less destabilizing groups, including G-G and G-T base mismatches, even when it locates very near the end of the probe.