Effect of thiazole orange doubly labeled thymidine on DNA duplex formation

Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ～ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/ . It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.     

Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

Novel non-isotopic detection of MutY enzyme-recognized mismatches in DNA via ultrasensitive detection of aldehydes.

A highly sensitive method to detect traces of aldehyde-containing apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based on this method, a novel approach to detect DNA base mismatches recognized by the mismatch repair glycosylase MutY is demonstrated. Open chain aldehydes generated in nucleic acids due to spontaneous depurination, DNA damage or base excision of mismatched adenine by MutY are covalently trapped by a new linker molecule [fluorescent aldehyde-reactive probe (FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing AP sites is FARP-trapped, biotinylated and immobilized onto neutravidin-coated microplates. The number of FARP-trapped aldehydes is then determined via chemiluminescence using a cooled ICCD camera. AP sites induced in plasmid or genomic calf thymus DNA via mild depurination or by simple incubation at physiological conditions (pH 7, 37 degreesC) presented a linear increase in chemiluminescence signal with time. The procedure developed, from a starting DNA material of approximately 100 ng, allows detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7) bases, and extends by 1-2 orders of magnitude the current limit in AP site detection. In order to detect MutY-recognized mismatches, nucleic acids are first treated with 5 mM hydroxylamine to remove traces of spontaneous aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides engineered to have a centrally located A/G mismatch demonstrate a strong chemiluminescence signal. Similarly, single-stranded M13 DNA that forms mismatches via self-complementation (average of 3 mismatches over 7429 bases) and treated with MutY yields a signal approximately 100-fold above background. No signal was detected when DNA without mismatches was used. The current development allows sensitive, non-isotopic, high throughput screening of diverse nucleic acids for AP sites and mismatches in a microplate-based format.     

Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays

DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific. Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.     

Gelonin is an unusual DNA glycosylase that removes adenine from single-stranded DNA, normal base pairs and mismatches

We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216-17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and beta-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k(cat) for the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant.     

An improved chemico-enzymatic method of synthesizing long DNA segments

A simple and economy method of the biochemical assembling of long double-stranded DNA segments is described. A single-stranded polydeoxynucleotide 122 bases long representing a fragment of synthetic gene of human beta-interferon was assembled from three synthetic fragments 36 (two) and 50 bases long on four complementary 12-mers as templates. This single-stranded polynucleotide was converted, in the presence of DNA polymerase 1 and a 12-meric primer, in to the full-length double-stranded DNA (the beta-interferon gene segment). It was cloned into an E. coli plasmid vector pBR322 and its sequence confirmed.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.     

Joining of short DNA oligonucleotides with base pair mismatches by T4 DNA ligase

Oligonucleotide-directed mutagenesis is a widely used method for studying enzymes and improving their properties. The number of mutants that can be obtained with this method is limited by the number of synthetic 25-30mer oligonucleotides containing the mutation mismatch, becoming impracticably large with increasing size of a mutant library. To make this approach more practical, shorter mismatching oligonucleotides (7-12mer) might be employed. However, the introduction of these oligonucleotides in dsDNA poses the problem of sealing a DNA nick containing 5'-terminal base pair mismatches. In the present work we studied the ability of T4 DNA ligase to catalyze this reaction. It was found that T4 DNA ligase effectively joins short oligonucleotides, yielding dsDNA containing up to five adjacent mismatches. The end-joining rate of mismatching oligonucleotides is limited by the formation of the phosphodiester bond, decreasing with an increase in the number of mismatching base pairs at the 5'-end of the oligonucleotide substrate. However, in the case of a 3 bp mismatch, the rate is higher than that obtained with a 2 bp mismatch. Increasing the matching length with the number of mismatching base pairs fixed, or moving the mismatching motif downstream with respect to the joining site increases the rate of ligation. The ligation rate increases with the molar ratio [oligonucleotide:dsDNA]; however, at high excess of the oligonucleotide, inhibition of joining was observed. In conclusion, 9mer oligonucleotides containing a 3 bp mismatch are found optimal substrates to introduce mutations in dsDNA, opening perspectives for the application of T4 DNA ligase in mutagenesis protocols.     

Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis

RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide- and RNase H-dependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.     

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.     

A New Approach to Determine the Effect of Mismatches on Kinetic Parameters in DNA Hybridization Using an Optical Biosensor

We have demonstrated a simple yet direct method for determiningthe kinetic parameters in DNA-DNA interactions using biosensortechnology based on the surface plasmon resonance phenomenon;a technique that does not require complex DNA labeling. To determinethe effect of mismatches on the kinetics involved in DNA-DNAinteractions, DNA hybridization kinetics were monitored in realtime using synthetic oligonucleotides less than 20 bases inlength which contained either a complementary sequence or mismatchedbases. Upon analysis of the kinetic parameters obtained in oligonucleotidehybridization, we found that they were significantly affectedby the presence of mismatches as well as by their number andlocation in a DNA duplex. In addition, the presented biosensormethod is sensitive enough to detect kinetic effects causedby the presence of a single-mismatched base pair. Our findingsstrongly suggest that analysis of kinetic parameters involvedin DNA-DNA interactions is advantageous for detecting the presenceof mismatch base pairs in a DNA duplex.     

DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe

We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm.     

Voltammetric detection of single base-pair mismatches and quantification of label-free target ssDNA using a competitive binding assay

The application of electrochemical techniques for DNA detection is motivated by their potential to detect hybridisation events in a more rapid, simplistic and cost-effective manner compared to conventional optical assays. Here, we present an electrochemical DNA sensor for the specific and quantitative detection of single-stranded DNA (ssDNA). Probe oligonucleotides were immobilised onto thin gold film electrodes by a 5'-thiol-linker. Hybridisation was detected by means of the electroactive redox-marker methylene blue (MB) covalently attached to the 5'-end of the target ssDNA and voltammetric techniques. MB-labeled target ssDNA was recognised down to 30 pmol. By application of a competitive binding assay, non-labeled ssDNA was detected down to 3 pmol. In addition, the DNA-modified electrodes were capable of sensing single base-pair mismatches at different positions within the sequence of the hybridised double-stranded DNA (dsDNA).     

Real-time oligonucleotide hybridization kinetics monitored by resonant mirror technique

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.     

Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides

The repair of point mutations in hprt gene by single-stranded oligonucleotides represents a model to test targeted nucleotide exchange. We studied the concurrent nucleotide exchange of two or three nucleotides in the hprt deficient hamster cell line V79-151. The used oligonucleotides resulted in mismatches at two (151, 159) or three (151, 144, and 159) hprt positions. The hprt point mutation at position 151 was repaired in about 2/10(6) cells as shown by hprt sequencing in clones surviving HAT selection. The second nucleotide exchange at hprt position 159 was found in 7% of these HAT selected clones. Using oligonucleotides resulting in three mismatches, 29% of the clones showed nucleotide exchanges at the two hprt positions (151, 144) and about 4% at three positions (151, 144, and 159). These results indicate that single-stranded oligonucleotides can generate two or three nucleotide exchanges in a mammalian chromosomal gene.     

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA

Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.     

Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes.

Oligonucleotide dendrimers were synthesized using a novel phosphoramidite synthon, tris-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyloxymethyl]ethyl- N , N -diisopropylaminocyanethoxy phosphoramidite. Label, incorporated using [gamma-32P]ATP and polynucleotide kinase, was increased in proportion to the number of 5'-ends. There was a similar increase in signal when these multiply labelled oligonucleotides were used as probes to oligonucleotide arrays. A dendrimeric oligonucleotide was used successfully as a primer in the PCR. The strand bearing the dendrimer was resistant to degradation by T7 Gene 6 exonuclease making it easy to convert the double-stranded product of the PCR to a multiply-labelled, single-stranded probe.     

Single nucleotide mismatch analysis using oligonucleotide probes synthesized on bacterial magnetic particle

An approach to analyze mismatches using short and specific oligonucleotide probes directly synthesized on bacterial magnetic particles (BMPs) by phosphoramidite methods was exploited. Approximately 126 molecules of 4-mer oligonucleotides/particle were synthesized on BMPs with high reaction efficiencies. Hybridization between FITC-labeled oligonucleotides and chemically synthesized oligonucleotides on BMPs was performed. Perfect matched and mismatched hybridizations were successfully discriminated by using the oligonucleotide probes on BMPs.