Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

A tolerance of DNA heterology in the mammalian targeted gene repair reaction

Targeted gene repair consists of at least two major steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. In this study, oligonucleotides with different structures were designed to target a stably integrated (mutant) enhanced green fluorescent protein (EGFP) gene and used to direct the repair of a single base mutation. We show that the efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. Correction is reduced when a heterologous stretch of DNA sequence is placed in the center of the oligonucleotide and the mismatched base pair is then formed near the terminus. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. If the heterologous sequence is palindromic and adopts a secondary structure, the negative impact on the correction frequency is removed, and wild-type levels of repair are restored. Although differences in the efficiency of correction are observed in various cell types, the effect of structural changes on gene repair is consistent. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Development of a simple coarse-grained DNA model for analysis of oligonucleotide complex formation

In this paper, we develop a coarse-grained nucleotide model for the purpose of simulating large-scale aptamer-based hydrogel network formation in future research. In the model, each nucleotide is represented by a single interaction site containing sugar, phosphate and base. Discontinuous molecular dynamics (DMD) simulations are performed to simulate formation and denaturation of oligonucleotide duplexes as a function of temperature. The simulated melting temperatures of oligonucleotide duplexes are calculated in simulations of systems with different sequences, lengths and concentrations of oligonucleotides, and compared to data from the OligoAnalyzer tool. The denaturation of oligonucleotide triplexes containing a hybridised structure of three different oligonucleotides is analysed using both simulations and experiments. The nucleotide model is found to be a good predictor of the oligonucleotide’s hybridised state for both duplexes and triplexes. This coarse-grained model has wide ranging applications in the development or optimisation of DNA-based technologies including DNA origami, DNA-enabled hydrogels and DNA-based biosensors.     

Parallel-stranded DNA with Hoogsteen base pairing stabilized by a trans-[Pt(NH3)2]2+ cross-link: characterization and conversion into a homodimer and a triplex

The oligonucleotides 5'-d(TTTTCTTTTG) and 5'-d(AAAAGAAAAG) were cross-linked with a trans-[Pt(NH3)2]2+ entity via the N7 positions of the 3'-end guanine bases to give parallel-stranded (ps) DNA. At pH 4.2, CD and NMR spectroscopy indicate the presence of Hoogsteen base pairing. In addition, temperature-dependent UV spectroscopy shows an increase in melting temperature for the platinated duplex (35 degrees C) as compared to the non-platinated, antiparallel-stranded duplex formed from the same oligonucleotides (21 degrees C). A monomer-dimer equilibrium for the platinated 20mer is revealed by gel electrophoresis. At pH 4.2, addition of a third strand of composition 5'-d(AGCTTTTCTTTTAG) to the ps duplex leads to the formation of a triple helix with two distinct melting points at 38 degrees C (platinum cross-linked Hoogsteen part) and 21 degrees C (Watson-Crick part), respectively.     

A statistical learning approach to the modeling of chromatographic retention of oligonucleotides incorporating sequence and secondary structure data

We propose a new model for predicting the retention time of oligonucleotides. The model is based on ν support vector regression using features derived from base sequence and predicted secondary structure of oligonucleotides. Because of the secondary structure information, the model is applicable even at relatively low temperatures where the secondary structure is not suppressed by thermal denaturing. This makes the prediction of oligonucleotide retention time for arbitrary temperatures possible, provided that the target temperature lies within the temperature range of the training data.

We describe different possibilities of feature calculation from base sequence and secondary structure, present the results and compare our model to existing models.

     

Optimizing the design of oligonucleotides for homology directed gene targeting

Background

Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit.

Methodology/Principal Findings

In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex.

Conclusion and Significance

A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.     

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.     

Sequence-targeted photochemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins

Porphyrins linked to oligonucleotides produce various types of photodamage on a complementary target DNA. The observed reactions include oxidation of guanine bases and cross-linking reactions of the oligonucleotide to its target sequence. Guanines located close to the porphyrin macrocycle were the most altered as compared to more remote guanines on the target sequence. No specific reaction was observed when the complexes were dissociated at temperatures above the melting temperature of the oligonucleotide-target hybrid. Both cross-linking and oxidation reactions accounted for ca. 60% modification of the target chains in the complex. Our results show that oligonucleotides covalently linked to porphyrins are efficient systems for inducing irreversible sequence-specific photodamage on a target DNA.     

Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site.

A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction.     

Formation of the double helix: a mutational study

To investigate the mechanisms by which oligonucleotides hybridize to target molecules, the binding of two oligodeoxynucleotide probes to RNA targets was measured over a broad range of temperatures. Mutations were then scanned across each DNA/RNA hybrid to map, at single base resolution, sequences important for hybridization. Despite being unrelated in sequence, each hybrid formed by a similar mechanism. In the absence of secondary structure, two stretches of bases, termed nucleation regions, cooperated with one another by a looping mechanism to nucleate hybridization. Mutations inside each nucleation region strongly decreased hybridization rates, even at temperatures well below the melting temperature (T_m) of the hybridized duplex. Surprisingly, nucleation regions were detected in a RNA target but not a corresponding DNA target. When either nucleation region was sequestered in secondary structure, the hybridization rate fell and the mechanism of hybridization changed. Single-stranded bases within the nucleation region of the probe and target first collided to form a double helix. If sufficiently G + C rich, the double helix then propagated throughout the oligonucleotide by a strand invasion process. On the basis of these results, general mechanisms for the hybridization of oligonucleotides to complementary and mutant targets are proposed.     

Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes

Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function we injected different miRNA-specific DNA antisense oligonucleotides in Drosophila embryos. In four cases we observed severe interference with normal development, one had a moderate impact and six oligonucleotides did not cause detectable phenotypes. We further used the miR-13a DNA antisense oligonucleotide as a PCR primer on a cDNA library template. In this experimental way we identified nine Drosophila genes, which are characterised by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirmed the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression.     

Triple helix formation inhibits DNA gyrase activity

The goal of this work was to examine the effect of triple helix-forming oligonucleotides on a gyrase target region and on the activity of the enzyme. Using melting temperature measurements and gel mobility shift analysis, it was found that modified oligonucleotides can form a triple helix along the 29-nucleotide region of a 32-bp duplex representing part of the gyrase DNA-target sequence of the 162-bp fragment from pBR322. Triplex formation with this target region has been achieved at pH 7.5 by using a synthetic oligonucleotide in which cytosine was replaced by the C-nucleoside of 2-aminopyridine. The results of the enzymic experiments in vitro with the 162-bp fragment demonstrated that the cleavage reaction mediated by gyrase can be efficiently inhibited by the triplex-forming oligonucleotide modified with 2-aminopyridine. A possible inhibitory mechanism is discussed.     

Analysis of perfect and mismatched DNA duplexes by a generic hexanucleotide microchip

The thermodynamic analysis was done for the duplexes formed by fluorescently labeled oligonucleotide targets on a genetic hexanucleotide microchip. All 4096 different hexanucleotide chains were immobilized as probes in individual gel pads of the microchip. To strengthen the hybridization, each hexamer was extended at both ends by one nucleotide from the equimolar mixture of all four nucleotides to serve as nonselective linkers. It has been shown that the melting curves for oligonucleotide duplexes formed on the microchip and in a solution are quite similar. The influence of ionic surrounding has been studied in terms of the hybridization efficiency and discrimination between the mismatched and perfect duplexes. Different approaches have been tested to compensate the dependence of duplex stability on the GC content. It has been demonstrated that the use of chaotropic agents, addition of nonlabeled GC-rich competitor oligonucleotides, as well as creation of a temperature gradient along the microchip reproducing the distribution of melting temperatures, efficiently level out the AT/GC differences. The use of tetramethylammonium chloride for the same purpose was accompanied by weakening to some extent the discrimination between the mismatched duplexes and the perfect ones.     

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: II. Applications to the analysis of subtle and drastic mobility changes of oligo- and polynucleotides.

Precise denaturing gradient gel electrophoresis was effectively applied to various kinds of oligo- and polynucleotides. The analyses on oligonucleotides revealed that every oligonucleotide has its own characteristic normalized mobility profile (NMP), which can be used to identify, characterize and classify the molecules. The precise system also enabled us to obtain unequivocally the mobility transitions corresponding to the melting of hairpin structures of oligonucleotides, single-stranded (ss) DNAs, and RNAs. Another application to co-migration and separate migration experiments demonstrated that there were significant binding interactions between two species of ss molecules of similar mobility, even when they have little complementarity with each other. When the precise temperature gradient gel electrophoresis was applied to double-stranded DNAs, it could be confirmed with high reliability that the mobility transitions observed correspond to cooperative meltings and strand dissociations. Through these experiments, mu m, a parameter defined as a mobility transition point, was shown to be effective to deal with those phenomena quantitatively.     

Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.

Arrays of oligonucleotides corresponding to a full set of complements of a known sequence can be made in a single series of base couplings in which each base in the complement is added in turn. Coupling is carried out on the surface of a solid support such as a glass plate, using a device which applies reagents in a defined area. The device is displaced by a fixed movement after each coupling reaction so that consecutive couplings overlap only a portion of previous ones. The shape and size of the device and the amount by which it is displaced at each step determines the length of the oligonucleotides. Certain shapes create arrays of oligonucleotides from mononucleotides up to a given length in a single series of couplings. The array is used in a hybridisation reaction to a labelled target sequence, and shows the hybridisation behaviour of every oligonucleotide in the target sequence with its complement in the array. Applications include sequence comparison to test for mutation, analysis of secondary structure, and optimisation of PCR primer and antisense oligonucleotide design.     

Analysis of Perfect and Mismatched DNA Duplexes with a Generic Hexanucleotide Microchip

Thermodynamic analysis was performed for the duplexes formed by fluorescently labeled oligonucleotide targets on a generic hexanucleotide microchip. All 4096 different hexanucleotide chains were immobilized as probes in individual gel pads of the microchip. To strengthen the hybridization, each hexamer was extended at both ends by one nucleotide from the equimolar mixture of all four nucleotides to serve as nonselective linkers. It has been shown that the melting curves for oligonucleotide duplexes formed on the microchip and in a solution are quite similar. The influence of ionic surrounding has been studied in terms of the hybridization efficiency and discrimination between the mismatched and perfect duplexes. Different approaches have been tested to compensate the dependence of duplex stability on the GC content. It has been demonstrated that the use of chaotropic agents, addition of nonlabeled GC-rich competitor oligonucleotides, as well as creation of a temperature gradient along the microchip reproducing the distribution of melting temperatures, efficiently level out the AT/GC differences. The use of tetramethylammonium chloride for the same purpose was accompanied by weakening to some extent the discrimination between the mismatched duplexes and the perfect ones.     

Reduction of gene repair by selenomethionine with the use of single-stranded oligonucleotides

Background  

The repair of single base mutations in mammalian genes can be directed by single-stranded oligonucleotides in a process known as targeted gene repair. The mechanism of this reaction is currently being elucidated but likely involves a pairing step in which the oligonucleotide align in homologous register with its target sequence and a correction step in which the mutant base is replaced by endogenous repair pathways. This process is regulated by the activity of various factors and proteins that either elevate or depress the frequency at which gene repair takes place.     

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.