Electrochemical evaluation of alternating duplex-triplex conversion effect on the anthraquinone-photoinjected hole transport through DNA duplex immobilized on a gold electrode

Amperometry was employed to characterize the anthraquinone (AQ)-photoinjected hole transport through a 20-mer oligodeoxynucleotide (ODN) duplex, as immobilized on the surface of a gold electrode, and its triplex forms converted by association with several third oligopyrimidine (OPD) short strands. While the cathodic photocurrent was observed upon irradiation at 365 nm of the AQ photosensitizer linked to the end of DNA duplex, a marked lowering of the current density was identified to occur by the triplex formation of a duplex with a given third OPD short strand. The photocurrent through the DNA duplex showed a reversible fall-rise response concomitant with alternating association-dissociation cycle of the OPD short-strand, as regulated by temperature change around the corresponding melting temperature of the DNA triplex. Both the switched photoirradiation and the thermally alternating duplex-triplex conversion could provide tools of regulating the DNA hole transport.     

A duplex–triplex nucleic acid nanomachine that probes pH changes inside living cells during apoptosis

A duplex–triplex switchable DNA nanomachine was fabricated and has been applied for the demonstration of intracellular acidification and apoptosis of Ramos cells, with graphene oxide (GO) not only as transporter but also as fluorescence quencher. The machine constructed with triplex-forming oligonucleotide exhibited duplex–triplex transition at different pH conditions. By virtue of the remarkable difference in affinity of GO with single-stranded DNA and triplex DNA, and the super fluorescence quenching efficiency of GO, the nanomachine functions as a pH sensor based on fluorescence resonance energy transfer. Moreover, taking advantage of the excellent transporter property of GO, the duplex–triplex/GO nanomachine was used to sense pH changes inside Ramos cells during apoptosis. Fluorescence images showed different results between living and apoptotic cells, illustrating the potential of DNA scaffolds responsive to more complex pH triggers in living systems.

Photocurrent response after enzymatic treatment of DNA duplexes immobilized on gold electrodes: electrochemical discrimination of 5-methylcytosine modification in DNA

We demonstrate a photoelectrochemical approach to the detection of the methylation status of cytosine bases in DNA. We prepared anthraquinone (AQ) photosensitizer-tethered oligodeoxynucleotide (ODN) duplexes bearing 5-methylcytosine (mC) or the corresponding cytosine (C) at a restriction site of the ODN strand immobilized on gold electrodes, and measured their photocurrent responses arising from hole transport after enzymatic digestion. Treatment with HapII or HhaI of the duplexes bearing normal C led to strand cleavage, and the photosensitizer unit was eliminated from the ODN strand immobilized on the gold electrode, exclusively reducing the photocurrent density. With a similar treatment, the duplexes bearing mC showed higher photocurrent responses arising from hole transport through the duplex. This significant difference in the photocurrent response between mC and normal C residues in DNA on the gold electrodes is potentially applicable to the detection of mC modification in DNA.     

Photostimulated hole transport through a DNA duplex immobilized on a gold electrode

Photostimulated hole transport through DNA duplexes immobilized on gold electrodes has been investigated. By modifying a gold electrode with a DNA duplex containing a photosensitizer, we have observed a sequence-dependent cathodic photocurrent. DNA acts as a good mediator for cathodic photocurrent when appropriate sequences are selected.     

Hairpin Probe for Sequence-specific Recognition of Double-stranded DNA on Simian Virus 40

Simian virus 40(SV40) is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine·homopurine duplex DNA(dsDNA) of SV40(4424—4440, gp6) was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5'-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3'(HP) containing G-rich sequence and 17-bp triplex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structure to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.     

Guanine of the third strand of C.G*G triplex serves as an effective hole trap

We have examined the structural and electronic effects of the one-electron oxidation of the C.GG triplex, where G is located in a quite different environment from the G of duplex DNA. Upon photoirradiation of an external photosensitizer (riboflavin) with the C.GG triplex, oxidative DNA cleavage occurred exclusively at guanine repeat sequences in the third strand of triple helix DNA. Hole transport through the C.GG triplex also occurred, resulting in selective cleavage at G in the third strand. Thus, the hole generated in the duplex can migrate to GGG in the third strand and is trapped exclusively at Gs in the third strand. These experimental results, together with molecular orbital calculations, suggest that the origin of the selective strand cleavage can be explained as follows: (i) guanine repeat sequences in the third strand are more easily oxidized than in duplex DNA and (ii) in their radical cation states, G of the third strand rapidly deprotonates and reacts with oxygen and/or water, leading to strand cleavage. These results indicate that the oxidative damage preferentially occurred at Gs of the third strand owing to thermodynamic and kinetic features of the one-electron oxidation of the C.GG triplex.     

DNA hole transport on an electrode: application to effective photoelectrochemical SNP typing

A useful feature of DNA is that long-range hole transport through DNA is readily achieved. Photostimulated long-range hole transport through DNA has prospective use in the development of a conceptually new electrochemical single-nucleotide polymorphism (SNP) typing method for use as a versatile platform for gene diagnostics and pharmacogenetics. We have applied artificial DNAs designed for photostimulated long-range hole transport through DNA to SNP typing. By hybridizing photosensitizer-equipped DNA probes, immobilized on gold working electrodes, with a target DNA strand containing an SNP site, we observed a cathodic photocurrent, which markedly changed depending on the nature of the base at the specific site. The use of a combination of hole-transporting bases constitutes a very powerful method for a single-step electrochemical assay applicable to SNP typing of all types of sequences.     

Fluorescently Sensing of DNA Triplex Assembly Using an Isoquinoline Alkaloid as Selector,Stabilizer, Inducer,and Switch‐On Emitter

DNA triplex assembly has attracted a variety of interest in the regulation of genetic expression, drug screening, molecular switches, and sensors. However, these achievements are essentially dependent on the formation and stability of the triplex assembly. Herein, the recognition of DNA triplex assembly with various isoquinoline alkaloids was investigated. We found that natural chelerythrine (CHE) exhibits the highest selectivity in recognizing the triplex structure. The DNA triplex stability is substantially increased upon CHE binding, as opposed to the invariance in the stability of the duplex counterpart. CHE also favors the assembly of the triplex‐forming oligonucleotide (TFO) with its duplex counterpart. The triplex binding switches CHE to a strong fluorescent emitter, which suggests CHE as a useful probe in following triplex assembly. As a unique triplex selector, inducer, and emitter, CHE successfully reports the wide pH‐ and metal‐ion‐dependent tunability of the triplex nanoswitch in a label‐free manner.     

Cleavage of oligonucleotides containing a P3'→N5' phosphoramidate linkage mediated by single-stranded oligonucleotide templates

Double-stranded DNA (dsDNA) templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3'→N5' phosphoramidate (P-N) linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.     

pH-responsive and switchable triplex-based DNA hydrogels

New methods for the preparation of reversible pH-responsive DNA hydrogels based on Hoogsteen triplex structures are described. One system consists of a hydrogel composed of duplex DNA units that bridge acrylamide chains at pH = 7.4 and undergoes dissolution at pH = 5.0 through the reconfiguration of one of the duplex bridging units into a protonated CG·C⁺ triplex structure. The second system consists of a hydrogel consisting of acrylamide chains crosslinked in the presence of an auxiliary strand by Hoogsteen TA·T triplex interaction at pH = 7.0. The hydrogel transforms into a liquid phase at pH = 10.0 due to the separation of the triplex bridging units. The two hydrogel systems undergo reversible and cyclic hydrogel/solution transitions by subjecting the systems to appropriate pH values. The anti-cancer drug, coralyne, binds specifically to the TA·T triplex-crosslinked hydrogel thereby increasing its stiffness. The pH-controlled release of the coralyne from the hydrogel is demonstrated.     

pH-independent triple-helix formation with 6-oxocytidine as cytidine analogue

The syntheses of six different phosphoramidite building blocks of 6-oxocytosine and 5-allyl-6-oxocytosine as analogues of N(3)-protonated cytosine are described. These compounds have been incorporated into oligonucleotides by standard solid-phase synthesis. Hybridization of 15-mer Hoogsteen strands with target 21-mer duplexes was investigated. Comparison of the triplex-forming abilities of the different building blocks revealed that: i) 5-allyl substitution has a negative influence on triplex stability, ii) a uniform backbone of the Hoogsteen strand stabilizes triplexes relative to mixed backbones; iii) RNA strands with 6-oxocytidine or 5-allyl-6-oxocytidine do not form a triple helix with the DNA target duplex, probably due to backbone torsional constraints; and (iv) a 15-mer DNA sequence with three isolated 2'-deoxy-6-oxocytidines has the highest T(m) of all cytidine analogues investigated in this study. CD experiments provided further evidence for the presence or absence of triplex structures. In the course of these temperature-dependent CD measurements we were able to detect duplex and triplex melting independent from each other at selected wavelengths. This methodology is especially interesting in cases where UV melting curves show only one transition owing to spectral overlap.     

Dynamics and energetics of single-step hole transport in DNA hairpins

The dynamics of single-step hole transport processes have been investigated in a number of DNA conjugates possessing a stilbenedicarboxamide electron acceptor, a guanine primary donor, and several secondary donors. Rate constants for both forward and return hole transport between the primary and secondary donor are obtained from kinetic modeling of the nanosecond transient absorption decay profiles of the stilbene anion radical. The kinetic model requires that the hole be localized on either the primary or the secondary donor and not delocalized over both the primary and the secondary donor. Rate constants for hole transport are found to be dependent upon the identity of the secondary donor, the intervening bases, and the location of the secondary donor in the same strand as the primary donor or in the complementary strand. Rate constants for hole transport are much slower than those for the superexchange process used to inject the hole on the primary donor. This difference is attributed to the larger solvent reorganization energy for charge transport versus charge separation. The hole transport rate constants obtained in these experiments are consistent with experimental data for single-step hole transport from other transient absorption studies. Their relevance to long-distance hole migration over tens of base pairs remains to be determined. The forward and return hole transport rate constants provide equilibrium constants and free energies for hole transport equilibria. Secondary GG and GGG donors are found to form very shallow hole traps, whereas the nucleobase deazaguanine forms a relatively deep hole trap. This conclusion is in accord with selected strand cleavage data and thus appears to be representative of the behavior of holes in duplex DNA. Our results are discussed in the context of current theoretical models of hole transport in DNA.     

Conformationally restricted triplex-forming oligonucleotides (TFOs). Binding properties of α-l-LNA and introduction of the N-glycosylated LNA-guanosine

The method for scaled-up production of α-l-LNA phosphoramidite building blocks containing thymine and 5-methylcytosine nucleobases is described. Binding properties of pyrimidine TFOs modified with α-l-LNA are reported. In contrast to LNA TFOs, the fully modified α-l-LNA forms a stable triplex with a model DNA duplex. Pyrimidine DNA/LNA/α-l-LNA chimeras also efficiently hybridize with a model DNA duplex in the parallel mode. LNA nucleoside containing unnatural N⁷-glycosylated guanine (LNA-⁷G) was synthesized by a convergent method and incorporated into LNA oligonucleotides. The triplex-forming alternating DNA/LNA oligonucleotides containing a single LNA-⁷G modification instead of internal LNA-mC demonstrate improved pH-dependent properties. The single LNA-⁷G modification can also discriminatively reduce competitive binding of TFOs to natural nucleic acids in the antiparallel duplex mode.     

Selective recognition of CG interruption by 2′,4′-BNA having 1-isoquinolone as a nucleobase in a pyrimidine motif triplex formation

To develop a novel nucleoside analogue for the effective recognition of CG interruption in a homopurine-homopyrimidine tract of double-stranded DNA (dsDNA), we succeeded in the synthesis of a triplex-forming oligonucleotide (TFO) containing a novel 2′,4′-BNA (Q^B) bearing 1-isoquinolone as a nucleobase, and the triplex-forming ability and sequence-selectivity of the TFO (TFO-Q^B) were examined. On melting temperature (T_m) measurements, it was found that the TFO-Q^B formed a stable triplex DNA in a highly sequence-selective manner under near physiological conditions.     

Neomycin binding to Watson-Hoogsteen (W-H) DNA triplex groove: a model

Neomycin is the most effective aminoglycoside (groove binder) in stabilizing a DNA triple helix. It stabilizes TAT, as well as mixed base DNA triplexes, better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (in the presence of salt), without any effect on the DNA duplex. (1) Triplex stabilization by neomycin is salt dependent (increased KCl and MgCl(2) concentrations decrease neomycin's effectiveness, at a fixed drug concentration). (2) Triplex stabilization by neomycin is pH dependent (increased pH decreases neomycin's effectiveness, at a fixed drug concentration). (3) CD binding studies indicate approximately 5-7 base triplets/drug apparent binding site, depending upon the structure/sequence of the triplex. (4) Neomycin shows nonintercalative groove binding to the DNA triplex, as evident from viscometric studies. (5) Neomycin shows a preference for stabilization of TAT triplets but can also accommodate CGC(+) triplets. (6) Isothermal titration calorimetry (ITC) studies reveal an association constant of approximately 2 x 10(5) M(-)(1) between neomycin and an intramolecular triplex and a higher K(a) for polydA.2polydT. (7) Binding/modeling studies show a marked preference for neomycin binding to the larger W-H groove. Ring I/II amino groups and ring IV amines are proposed to be involved in the recognition process. (8) The novel selectivity of neomycin is suggested to be a function of its charge and shape complementarity to the triplex W-H groove, making neomycin the first molecule that selectively recognizes a triplex groove over a duplex groove.     

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.     

Development of bridged nucleic acid analogues for antigene technology

In the last decade, increased efforts have been directed toward the development of oligonucleotide-based technologies for genome analyses, diagnostics, or therapeutics. Among them, an antigene strategy is one promising technology to regulate gene expression in living cells. Stable triplex formation between the triplex-forming oligonucleotide (TFO) and the target double-stranded DNA (dsDNA) is fundamental to the antigene strategy. However, there are two major drawbacks in triplex formation by a natural TFO: low stability of the triplex and limitations of the target DNA sequence. To overcome these problems, we have developed various bridged nucleic acids (BNAs), and found that the 2',4'-BNA modification of oligonucleotides strongly promotes parallel motif triplex formation under physiological conditions. Some nucleobase analogues to extend the target DNA sequence were designed, synthesized, and introduced into the 2',4'-BNA structure. The obtained 2',4'-BNA derivatives with unnatural nucleobases effectively recognized a pyrimidine-purine interruption in the target dsDNA. Some other examples of nucleic acid analogues for stable triplex formation and extension of the target DNA sequence are also summarized.     

Stable oligonucleotide-directed triplex formation at target sites with CG interruptions: strong sequence-specific recognition by 2',4'-bridged nucleic-acid-containing 2-pyridones under physiological conditions

A sequence of double-stranded DNA (dsDNA) which can be recognized by a triplex-forming oligonucleotide (TFO) is limited to a homopurine-homopyrimidine sequence. To develop novel nucleoside analogues which recognize CG interruption in homopurine-homopyrimidine dsDNA, we synthesized a novel 2'-O,4'-C-methyleneribonucleic acid (2'-O,4'-C-methylene bridged nucleic acid; 2',4'-BNA) that bears the unnatural nucleobases, 2-pyridone (PB) or its 5-methyl congener (mPB); these analogues were introduced into pyrimidine TFOs using a DNA synthesizer. A TFO with a 2'-deoxy-beta-D-ribofuranosyl-2-pyridone (P) or 2',4'-BNA abasic monomer (HB) was also synthesized. The triplex-forming ability of various synthesized 15-mer TFOs and the corresponding homopurine-homopyrimidine dsDNA, which contained a single pyrimidine-purine (PyPu) interruption, was examined in UV melting experiments. It was found that PB and mPB in the TFOs successfully recognized CG interruption under physiological conditions (7 mM sodium phosphate, 140 mM KCl, 5 mM spermine, pH 7.0). Furthermore, triplex formation between the dsDNA target which contained three CG interruptions and the TFO with three PB units was also confirmed. Additional four-point 2',4'-BNA modifications of the TFO containing three PB units significantly enhanced its triplex-forming ability towards the dsDNA and had a Tm value of 43 degrees C under physiological conditions. These results indicate that a critical inherent problem of TFOs, namely, the sequence limitation of the dsDNA target, may be overcome to a large extent and this should promote antigene applications of TFOs in vitro and in vivo.     

Rupture force between the third strand and the double strand within a triplex DNA

The rupture force to separate the third strand and the duplex within a triplex DNA was measured by means of atomic force spectroscopy. The tip and the sample surfaces were functionalized by oligodeoxyribonucleotides 5'-TTCTTCTTTCTTTTCCTTTTCTTTCTTCTTACTTCTCTCTCTC TCTCTCT-SH-3'. The sample surface was hybridized with 5'-AAGAAGAAAGAAAAGGAAAAGAAAGAAGAA-3' to form a double strand DNA on the surface prior to the force measurements. These sequences form triple helices with 30 base pairs under a pH of 5.8 and in the presence of 2.0 mM spermine. Signals of rupture of single and multiple triplex DNA were observed in the force distance curves. Rupture force histograms revealed a force of 42.6 +/- 1.9 pN from 24 independent measurements at a tip velocity of 400 nm/s to separate the third strand from duplex DNA. The velocity dependence of the rupture force quantum indicates a thermal dissociation process similar to that of rupturing a ds-DNA. The number of rupture events was controlled by adding oligonucleotides 5'-AAGAAGAAAGAAAAGGAAAAGAAAGAAGAA-3' either to reduce or to initiate triplex formation.     

Synthesis and DNA duplex recognition of a triplex-forming oligonucleotide with an ureido-substituted 4-phenylimidazole nucleoside

A 4-(3-n-butylureidophenyl)imidazole nucleoside was successfully incorporated into a triplex-forming oligonucleotide (TFO). Binding affinity and base pair selectivity of the TFO containing this non-natural nucleoside were studied with various duplex targets containing all four possible Watson-Crick base pairs opposite the nucleoside analog in the third strand. Triplex thermal stabilities indicate that the synthetic nucleoside acts as a universal base in binding to all four possible Watson-Crick base pairs with moderate affinity but poor selectivity. Based on an analysis of its binding thermodynamics, this can be rationalized by the absence of strong specific interactions and more favorable entropic contributions upon triplex formation.