RNase HII from Chlamydia pneumoniae discriminates mismatches incorporation into DNA-rN1-DNA/DNA duplexes

It was reported that RNase HII from Chlamydia pneumoniae (CpRNase HII) had RNase H activity on RNA/DNA duplex. We have analyzed the cleavage specificity of CpRNase HII on DNA-rN1-DNA/DNA duplex (rN1, one ribonucleotide). Various mismatches were introduced into the DNA-rN1-DNA/DNA duplexes at or around the ribonucleotide. The mismatches of duplexes resulted in slower cleavage rates compared to the matched duplexes. Furthermore, a greater reduction in cleavage activity was observed for the mismatches located at or adjacent to the ribonucleotide. The mismatches at the same position of DNA-rN1-DNA/DNA duplexes have different impact on the cleavage rates of CpRNase HII depending on the types of mismatches. These findings may offer further insights into the physical binding and catalytic properties of CpRNase HII-substrate interaction.     

Effect of the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin on proofreading by Escherichia coli DNA polymerase I (Klenow fragment) in different sequence contexts

Oxidative damage to DNA by endogenous and exogenous reactive oxygen species has been directly linked to cancer, aging, and a variety of neurological disorders. The potential mutagenicity of the primary guanine oxidation product 8-oxo-7,8-dihydroguanine (Og) has been studied intensively, and much information is available about its miscoding potential in vitro and in vivo. Recently, a variety of DNA lesions have been identified as oxidation products of both guanine and 8-oxoguanine, among them spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). To address questions concerning the mutagenic potential of these secondary products of guanine oxidation, the effect of the lesions on proofreading by DNA polymerase was studied in vitro using the Klenow fragment of Escherichia coli polymerase I (Kf exo+). For the first time, k(cat)/K(m) values were obtained for proofreading of the X:N mismatches (X = Og, Gh, or Sp; N = A, G, or C). Proofreading studies of the terminal mismatches demonstrated the significance of the sequence context flanking the lesion on the 3' side. In addition, a sequence dependence was observed for Gh based on the identity of the base on the 5' side of the lesion providing evidence for a primer slippage mode if N was complementary to the 5' base. Internal mismatches were recognized by Kf exo+ resulting in the excision of the correct base pairs flanking mismatches from the 5' side. The absence of a sequence effect for the Gh- and Sp-containing duplexes can be attributed to the severe destabilization of the lesion-containing duplexes that promotes interaction with the exonuclease domain of the Klenow fragment.     

Very stable mismatch duplexes: structural and thermodynamic studies on tandem G.A mismatches in DNA.

We have used ultraviolet melting techniques to compare the stability of several DNA duplexes containing tandem G.A mismatches to similar duplexes containing tandem A.G, I.A, and T.A base pairs. We have found that tandem G.A mismatches in 5'-Y-G-A-R-3' duplexes are more stable than their I.A counterparts and that they are sometimes more stable than tandem 5'-Y-T-A-R-3' sequences. This is not the case for tandem G.A mismatches in other base stacking environments, and it suggests that tandem G.A mismatches in 5'-Y-G-A-R-3' sequences have a unique configuration. In contrast to tandem 5'-G-A-3' mismatches, tandem 5'-A-G-3' mismatches were found to be unstable in all cases examined.     

Chemical interrogation of mismatches in DNA-DNA and DNA-RNA duplexes under nonstringent conditions by selective 2'-amine acylation

2'-Amine-substituted nucleotides in hybridized duplexes can be chemically tagged in an acylation reaction that is faster for mismatched or flexible nucleotides than for residues constrained by base pairing. Here we explore mismatch and hybridization detection using probe oligodeoxynucleotides containing single 2'-aminocytidine or -uridine nucleotides annealed to DNA or RNA targets under nonstringent conditions, below T(m). Consistent with a mechanism in which 2'-amine acylation is gated by local nucleotide flexibility, we find that efficient acylation is correlated with formation of weaker or fewer hydrogen bonds in base pair mismatches. Using 2'-aminocytidine-containing probes annealed to both DNA and RNA targets, mismatches are reliably detected as rapid selective acylation of the 2'-amine group in two sequence contexts. For probe oligonucleotides containing 2'-aminouridine residues, good discrimination between U-A base pairs and U-G mismatches could be obtained for DNA-DNA but not for DNA-RNA duplexes upon the introduction of a single 2'-O-Me group 5' to the 2'-amino nucleotide. The 2'-O-Me group introduces a structural perturbation, presumably to a more A-form-like structure, that exaggerates local flexibility at mismatches in DNA strands. Thus, 2'-amine acylation can be used to interrogate all possible mismatches in DNA-DNA duplexes and mismatches involving 2'-amine-substituted cytidine nucleotides in DNA-RNA heteroduplexes. Applications of this chemistry include detecting and chemically proofreading single nucleotide polymorphisms in both DNA and RNA targets and quantifying absolute amounts of RNA.     

Thermodynamics of single mismatches in RNA duplexes

The thermodynamic properties and structures of single mismatches in short RNA duplexes were studied in optical melting and imino proton NMR experiments. The free energy increments at 37 degrees C measured for non-GU single mismatches range from -2.6 to 1.7 kcal/mol. These increments depend on the identity of the mismatch, adjacent base pairs, and the position in the helix. UU and AA mismatches are more stable close to a helix end, but GG mismatch stability is essentially unaffected by the position in the helix. Approximations are suggested for predicting stabilities of single mismatches in short RNA duplexes.     

Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses.

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.     

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.     

Conformational determinants of tandem GU mismatches in RNA: insights from molecular dynamics simulations and quantum mechanical calculations

Structure and energetic properties of base pair mismatches in duplex RNA have been the focus of numerous investigations due to their role in many important biological functions. Such efforts have contributed to the development of models for secondary structure prediction of RNA, including the nearest-neighbor model. In RNA duplexes containing GU mismatches, 5'-GU-3' tandem mismatches have a different thermodynamic stability than 5'-UG-3' mismatches. In addition, 5'-GU-3' mismatches in some sequence contexts do not follow the nearest-neighbor model for stability. To characterize the underlying atomic forces that determine the structural and thermodynamic properties of GU tandem mismatches, molecular dynamics (MD) simulations were performed on a series of 5'-GU-3' and 5'-UG-3' duplexes in different sequence contexts. Overall, the MD-derived structural models agree well with experimental data, including local deviations in base step helicoidal parameters in the region of the GU mismatches and the model where duplex stability is associated with the pattern of GU hydrogen bonding. Further analysis of the simulations, validated by data from quantum mechanical calculations, suggests that the experimentally observed differences in thermodynamic stability are dominated by GG interstrand followed by GU intrastrand base stacking interactions that dictate the one versus two hydrogen bonding scenarios for the GU pairs. In addition, the inability of 5'-GU-3' mismatches in different sequence contexts to all fit into the nearest-neighbor model is indicated to be associated with interactions of the central four base pairs with the surrounding base pairs. The results emphasize the role of GG and GU stacking interactions on the structure and thermodynamics of GU mismatches in RNA.     

C5-(1-propynyl)-2'-deoxy-pyrimidines enhance mismatch penalties of DNA:RNA duplex formation

UV melting experiments show that C5-(1-propynyl)ation of seven pyrimidines to give a fully propynylated oligodeoxynucleotide (PrODN) heptamer increases the thermodynamic stability of six Watson-Crick paired DNA:RNA duplexes by 8.2 kcal/mol, on average, at 37 degrees C. About 2.5 kcal/mol of this enhancement is due to long-range cooperativity between the propynylated pyrimidines, Y(p)'s. On average, penalties for dU(p):rG, dC(p):rA, dU(p):rC, and dC(p):rC mismatches are enhanced by 2.9 kcal/mol in PrODN:RNA duplexes over those in unmodified duplexes. This results in penalties as large as 10 kcal/mol for a single mismatch. Removing a single propyne two base pairs away from a mismatch in a PrODN:RNA duplex eliminates the enhancement in specificity. Evidently, enhanced specificity is directly linked to long-range cooperativity between Y(p)'s. In most cases, the enhanced specificity is larger for internal than for terminal mismatches. PrODN:RNA duplexes are destabilized by full phosphorothioate backbone substitution to give S-PrODN:RNA duplexes. The S-PrODN:RNA duplexes retain enhanced mismatch penalties, however. These results provide insight for utilizing long-range cooperativity and enhanced specificity to improve nucleic acid based probe and drug design.     

Detection of terminal mismatches on DNA duplexes in homogeneous assays or with immobilized probes

We recently reported the design of new fluorescent oligo-2'-deoxyribonucleotides (FODNs) for the detection of terminal mismatches on DNA duplexes in homogeneous assays. We now report the validation of this method in homogeneous assays with other sequences and the feasibility of the detection of terminal mismatches with immobilized FODNs. In all cases studied, the mismatched duplexes were more fluorescent than the perfect ones and results confirmed that the discrimination factor is sequence-dependent.     

Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.     

Detection of Terminal Mismatches on DNA Duplexes in Homogeneous Assays or with Immobilized Probes

We recently reported the design of new fluorescent oligo-2′-deoxyribonucleotides (FODNs) for the detection of terminal mismatches on DNA duplexes in homogeneous assays. We now report the validation of this method in homogeneous assays with other sequences and the feasibility of the detection of terminal mismatches with immobilized FODNs. In all cases studied, the mismatched duplexes were more fluorescent than the perfect ones and results confirmed that the discrimination factor is sequence-dependent.     

Mutation detection by electrocatalysis at DNA-modified electrodes

Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3-. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and "hot spot" mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.     

A guanine-linked end-effect is a sensitive reporter of charge flow through DNA and RNA double helices

The property of charge (electron hole) flow in DNA duplexes has been the subject of intensive study. RNA-DNA heteroduplexes have also been investigated; however, little information exists on the conductive properties of purely RNA duplexes. In investigating the relative conductive properties of a three molecule DNA-DNA duplex design, using piperidine and aniline to break strands at modified bases, we observed that duplexes with guanine-rich termini generated a large oxidative end-effect, which could serve as a highly sensitive reporter of charge flow through the duplexes. The end-effect was found faithfully to report attenuations in charge flow due to certain single-base mismatches within a duplex. Comparative charge flow experiments on DNA-DNA and RNA-RNA duplexes found large end-effects from both, suggesting that the A and B family of double helices conduct charge comparably. The sheer magnitude of the end-effect, and its high sensitivity to helical imperfections, suggest that it may be exploited as a sensitive reporter for DNA mismatches, as well as a versatile device for studying the structure, folding, and dynamics of complexly folded RNAs and DNAs.     

Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element

RNA interference (RNAi) is a powerful method for suppressing the expression of a gene of interest, and can be induced by 21-25 nucleotide small interfering RNA (siRNA) duplexes homologous to the silenced gene, which function as sequence-specific RNAi mediators in RNA-induced silencing complexes (RISCs). In the previous study, it was shown that fork-siRNA duplexes, whose sense-stranded siRNA elements carried a few nucleotide mismatches at the 3'-ends against the antisense-stranded siRNA elements, could enhance RNAi activity more than conventional siRNA duplexes in cultured mammalian cells. In this study, we further characterized fork-siRNA duplexes using reporter plasmids carrying target sequences complementary to the sense- or antisense-stranded siRNA elements in the untranslated region of Renilla luciferase. The data presented here suggest that nucleotide mismatches at either the 3'- or 5'-end of the sense-stranded siRNA elements in fork-siRNA duplexes could influence assembly of not only the antisense-stranded siRNA elements but also the sense-stranded elements into RISCs. In addition, we further suggest the possibility that there could be a positional effect of siRNA duplex on RNAi activity.     

Thermodynamics and kinetics for base pair opening in the DNA decamer duplexes containing cyclobutane pyrimidine dimer

The cyclobutane pyrimidine dimer (CPD) is one of the major classes of cytotoxic and carcinogenic DNA photoproducts induced by UV light. Hydrogen exchange rates of the imino protons were measured for various CPD-containing DNA duplexes to better understand the mechanism for CPD recognition by XPC-hHR23B. The results here revealed that double T·G mismatches in a CPD lesion significantly destabilized six consecutive base pairs compared to other DNA duplexes. This flexibility in a DNA duplex caused at the CPD lesions with double T·G mismatches might be the key factor for damage recognition by XPC-hHR23B.     

Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.     

Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine.

The synthesis of a diaminopurine PNA monomer, N-[N6-(benzyloxycarbonyl)-2,6-diaminopurine-9-yl] acetyl-N-(2-t-butyloxycarbonylaminoethyl)glycine, and the incorporation of this monomer into PNA oligomers are described. Substitution of adenine by diaminopurine in PNA oligomers increased the T m of duplexes formed with complementary DNA, RNA or PNA by 2.5-6.5 degrees C per diaminopurine. Furthermore, discrimination against mismatches facing the diaminopurine in the hybridizing oligomer is improved. Finally, a homopurine decamer PNA containing six diaminopurines is shown to form a (gel shift) stable strand displacement complex with a target in a 246 bp double-stranded DNA fragment.     

Evaluating single-base-pair discriminating capability of planar oligonucleotide microchips using a non-equilibrium dissociation approach

The capability of planar rRNA-based oligonucleotide microarrays for single-base-pair discrimination was evaluated using an approach that compares the non-equilibrium dissociation profiles and dissociation temperatures (Tds) of all probe-target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the Td approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5-10 degrees C and 10-15 degrees C, respectively, higher than the Tds of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, Td, and single-base-pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.