Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T_m) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 10¹⁸ Ų), a constant probe density (5 × 10¹² molecules/cm²) and spacer length (15 dT). The authors found that T_m for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T_m decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Effects of base mismatches on joining of short oligodeoxynucleotides by DNA ligases.

The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets. Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases. Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers. The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer. Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction. In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers. The results are relevant to mechanisms of ligation.     

High sequence fidelity in a non-enzymatic DNA autoligation reaction.

The success of oligonucleotide ligation assays in probing specific sequences of DNA arises in large part from high enzymatic selectivity against base mismatches at the ligation junction. We describe here a study of the effect of mismatches on a new non-enzymatic, reagent-free method for ligation of oligonucleotides. In this approach, two oligonucleotides bound at adjacent sites on a complementary strand undergo autoligation by displacement of a 5'-end iodide with a 3'-phosphorothioate group. The data show that this ligation proceeds somewhat more slowly than ligation by T4 ligase, but with substantial discrimination against single base mismatches both at either side of the junction and a few nucleotides away within one of the oligonucleotide binding sites. Selectivities of >100-fold against a single mismatch are observed in the latter case. Experiments at varied concentrations and temperatures are carried out both with the autoligation of two adjacent linear oligonucleotides and with intramolecular autoligation to yield circular 'padlock' DNAs. Application of optimized conditions to discrim-ination of an H- ras codon 12 point mutation is demonstrated with a single-stranded short DNA target.     

Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase

We present cognate base pair selectivity in template‐dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds‐Pa. T4 DNA ligase efficiently recognizes the Ds‐Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base‐pair selectivity, relative to the hydrogen‐bond interactions between pairing bases. The efficient ligation using Ds‐Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi‐synthetic organisms containing UBPs.     

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.     

T4-DNA ligase: substrate properties of synthetic DNA-duplexes with structural anomalies

Structural defects, affecting T4 DNA ligase function, were revealed with the help of synthetic DNA duplexes, containing modifications at single nick. Changes of configuration at C2' and C3' atoms of furanose in the acceptor terminus lead to total blocking of the nick sealing activity of T4 DNA ligase. On the contrary, substitution of 3'-terminal deoxyribonucleotide for ribonucleotide doesn't affect the enzyme's action. The duplex looses all of it's substrate activity if the next from the nick G.C pair is substituted for the noncomplementary G.C pair. In DNA duplexes containing an unpaired base in the nick, elimination of the extrahelical nucleotide proceeds the ligation step. In these cases the duplex substrate activity decreases depending on the extent of extrahelical base stacking into the double stranded DNA.     

Direct probing: covalent attachment of probe DNA to double-stranded target DNA.

We report a novel procedure, which can be applied to probing of specific DNA, for covalently attaching probe DNA to complementary sequences in double-stranded target DNA. Employing hairpin-like oligonucleotide probes in combination with successive use of recA protein and DNA ligase, probes can be attached directly to target DNA molecules without dissociation of the DNA. The hairpin-like structure of the probes was designed so that the terminus of the probe oligonucleotide can be brought into close stereochemical proximity to the terminus of the complementary strand of target DNA for ligation. Because of the elimination of the DNA dissociation and subsequent hybridization (and washing) steps in the currently employed method, the probing process has become greatly simplified and more efficient and may lead to development of fully automated probing systems.     

Template-independent ligation of single-stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.     

Strand-invading linear probe combined with unmodified PNA

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40 °C at 100 mM NaCl concentration.     

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.     

Expression,purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg²⁺ and Mn²⁺. The optimal concentrations of ATP cofactor and Mg²⁺ ion were 0.01–2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5′ to the nick inhibited ligation more than those at the first position 3′ to the nick. The mismatches at other positions 5′ to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5′ to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5′ to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.     

Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than thermus thermophilus DNA ligase

To mimic large numbers of nicked DNA duplexes we used a technique that produces nicked duplex DNA substrates by hybridization of complementary oligonucleotides, adjacent to an initiating primer, which are ligated together by a thermostable DNA ligase. Sequential ligation of nonanucleotides to this primary duplex results in the formation of polymers that can be analyzed by gel electrophoresis. The extent of polymerization is a measure of the efficiency of ligation. We determined the efficiency of ligation of nonanucleotides, using various length initiating primers, with three thermostable DNA ligases: Thermus thermophilus (Tth), Thermus scotoductus (Ts), and Rhodothermus marinus (Rm). Analysis of the effect of temperature for each ligase, and for each directing primer length, revealed that at 37 and 41 degrees C there was variation between ligase efficiency in the order Rm > or = Ts > or = Tth. The higher temperature of 46 degrees C was optimal for polymerization with each of the ligases and Rm ligase was the most efficient. Analysis of directionality of the ligations reactions suggests that for each of the Thermus ligases we tested, there was a bias to polymerization of nonanucleotides in a 5'-3' direction.     

SYNTHESIS AND CHARACTERIZATION OF DNA DUPLEXES CONTAINING AN N3T-ETHYL-N3T INTERSTRAND CROSSLINK IN OPPOSITE ORIENTATIONS

DNA duplexes containing an ethyl interstrand crosslink that bridges the N³ atoms of thymidines on the opposite strands have been synthesized using an approach that combines conventional solid phase oligonucleotide synthesis and the selective removal of protecting groups of a crosslinked thymidine dimer. This approach allows for the assembly of a crosslinked duplex directly on the solid support. Duplexes that contain a N³T-ethyl-N³T interstrand crosslink in a staggered orientation at either a -TA- or -AT- step in a duplex have been prepared. When placed in an -AT- step of a duplex the effect was stabilizing relative to the non-crosslinked control duplex (ΔT_m=+24°C) and this crosslinked duplex was found to efficiently form multimers in the presence of T4 ligase. In the case of the -TA- crosslinked duplex the stabilizing effect was less pronounced (ΔT_m=+6°C) and likewise did not undergo self ligation under identical conditions. Molecular modeling studies suggested that the -AT- containing lesion had little deviation in structure relative to the non-crosslinked duplex DNA control, whereas the -TA- crosslinked duplex exhibited significant buckling of the base pairs flanking the lesion.     

An error-prone viral DNA ligase

Our recent demonstration that DNA polymerase X (Pol X), the DNA repair polymerase encoded by the African swine fever virus (ASFV), is extremely error prone during single-nucleotide gap filling led us to hypothesize that it might contribute to genetic variability in ASFV. For the infidelity of Pol X to be relevant, however, the DNA ligase working downstream of it would need to be capable of sealing nicks containing 3'-OH mismatches. We therefore examined the nick ligation capabilities of the ASFV-encoded DNA ligase and here report the first complete 3' fidelity analysis, employing catalytic parameters, for any DNA ligase. The catalytic efficiency of nick sealing by both ASFV DNA ligase and bacteriophage T4 DNA ligase was determined in the steady state for substrates containing all 16 possible matched and mismatched base pair combinations at the 3' side of a nick. Our results indicate that ASFV DNA ligase is the lowest-fidelity DNA ligase ever reported, capable of ligating a 3' C:T mismatched nick (where C and T are the templating and nascent nucleotides, respectively) more efficiently than nicks containing Watson-Crick base pairs. Comparison of the mismatch specificity of Pol X with that of ASFV DNA ligase suggests that the latter may have evolved toward low fidelity for the purpose of generating the broadest possible spectrum of sealed mismatches. These findings are discussed in light of the genetic and antigenic variability observed among some ASFV isolates. Two novel assays for determining the concentration of active DNA ligase are also reported.     

Gene cloning,purification, and characterization of recombinant DNA ligases of the thermophilic archaea <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis> and <Emphasis Type="Italic">Methanobacterium thermoautotrophicum</Emphasis>

DNA ligase genes of the thermophilic archaeae Pyrococcus abyssi (Pab DNA ligase) and Methanobacterium thermoautotrophicum (Mth DNA ligase) were cloned in Escherichia coli. The resulting recombinant enzymes were tested for activity in a ligation mixture with two oligonucleotides, one containing a preformed hairpin structure. The yield of the reaction products was maximal at temperatures close to 70°C for either enzyme; their accumulation reached a plateau at 70–75% of the theoretical yield at a stoichiometric enzyme-to-substrate ratio. The enzymes differed in thermal stability. The half-life of Pab DNA ligase was approximately 60 min at 90°C. Mth DNA ligase was completely inactivated within 10 min at this temperature. The recombinant DNA ligases from P. abyssi and M. thermoautotrophicum remained stabile during long-term storage at 4°C.     

Thermodynamic basis of selectivity in guide-target-mismatched RNA interference

Silencing in RNAi is strongly affected by guide‐strand/target‐mRNA mismatches. Target nucleation is thought to occur at positions 2–8 of the guide (“seed region”); successful hybridization in this region is the primary determinant of target‐binding affinity and hence target cleavage. To define a molecular basis for the target sequence selectivity in RNAi, we studied all possible distinct single mismatches in seven positions of the seed region—a total of 21 substitutions. We report results from soft‐core thermodynamic integration simulations to determine changes in targeting binding‐free energies to Argonaute due to single mismatches in the guide strand, which arise during binding of an imperfectly matched target mRNA. In agreement with experiment, most mismatches impair target binding, consistent with a prominent role for binding affinity changes in RNAi sequence selectivity. Individual Argonaute residues located near the mismatched base pair are found to contribute significantly to binding affinity changes. We also use this methodology to analyze the mismatch‐dependent free energy changes for dissociation of a DNA?RNA hybrid from Argonaute, as a model for the escape of miRNAs from the silencing pathway. Several mismatched sequences of the miRNA have increased affinity to Argonaute, implying that some mismatches may reduce the probability for escape. Furthermore, calculations of base‐substitution‐dependent free energy changes for binding ssDNA reveal mild sequence sensitivity as expected for guide strand binding to Argonaute. Our findings give a thermodynamic basis for RNAi target sequence selectivity and suggest that miRNA mismatches may increase silencing effectiveness and thus could be evolutionarily advantageous. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.     

A high-throughput assay for the comprehensive profiling of DNA ligase fidelity

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.     

Fidelity of enzymatic ligation for DNA computing.

We describe a convenient assay for rapid qualitative evaluation of hybridization/ligation fidelity. The approach uses randomized probe strands of DNA and restriction enzyme digestion after amplification of reaction products by the polymerase chain reaction (PCR). We report ligation efficiencies and fidelities of two DNA ligases, T4 DNA ligase and Thermus aquaticus (Taq) DNA ligase, over a range of temperatures.     

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs.