Pyrimidine Motif Triplex Research Articles

Interaction of isoquinoline alkaloids with pyrimidine motif triplex DNA by mass spectrometry and spectroscopies reveals diverse mechanisms

Isoquinoline alkaloids represent an important class of molecules due to their broad range of pharmacology and clinical utility. Prospective development and use of these alkaloids as effective anticancer agents have elicited great interest. In this study, in order to reveal structure-activity relationship, we present the characterization of bioactive isoquinoline alkaloid-DNA triplex interactions, with particular emphasis on the sequence selectivity and preference of binding to the two types of DNA triplexes, by electrospray ionization mass spectrometry (ESI-MS) and various spectroscopic techniques. The six alkaloids, including coptisine, columbamine, epiberberine, berberrubine, jateorhizine, and fangchinoline, were selected to explore their interactions with the TC and TTT triplex DNA structures. Berberrubine, fangchinoline, coptisine, columbamine, and epiberberine have preference for TC rich DNA sequences compared to TTT rich DNA triplex based on affinity values in MS. The experimental results from different fragmentation modes in tandem MS, subtractive and hyperchromic effects in UV absorption spectra, fluorescence quenching and enhancement in fluorescence spectra, and strong conformational changes in circular dichroism (CD) hinted that the interaction between isoquinoline alkaloid-TC/TTT DNA had diverse mechanisms including at least two different binding modes: the electrostatic binding and the intercalation binding. Interestingly, columbamine, berberrubine, and fangchinoline can stabilize TTT triplex as inferred from optical thermal melting profiles, while it was not the case in TC triplex. These results provide new insights into binding of isoquinoline alkaloids to pyrimidine motif triplex DNA.

2'-O,4'-C-ethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability under physiological condition

Since pyrimidine motif triplex DNA is unstable at physiological neutral pH, triplex stabilization at physiological neutral pH is important for improvement of its potential to be applied to various methods in vivo, such as repression of gene expression, mapping of genomic DNA and gene-targeted mutagenesis. For this purpose, we studied the thermodynamic and kinetic effects of a chemical modification, 2'-O,4'-C-ethylene bridged nucleic acid (ENA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological neutral pH. Thermodynamic investigations indicated that the modification achieved more than 10-fold increase in the binding constant of the triplex formation. The increased number of the modification in TFO enhanced the increased magnitude of the binding constant. On the basis of the obtained thermodynamic parameters, we suggested that the remarkably increased binding constant by the modification may result from the increased stiffness of TFO in the unbound state. Kinetic studies showed that the considerably decreased dissociation rate constant resulted in the observed increased binding constant by the modification. We conclude that ENA modification of TFO could be a useful chemical modification to promote the triplex formation under physiological neutral condition, and may advance various triplex formation-based methods in vivo.

Direct observation of the formation of DNA triplexes by single-molecule FRET measurements

In this report we investigated the effects of various biological and chemical factors (DNA sequence, pH, ions, and molecularity) on the formation of DNA triplexes through single-molecule FRET technique. Using this method, we determined how the third strand bound to a DNA duplex and how stable the triplex structure was under various conditions. From this study, we not only verified a variety of well-known features of DNA triplex but also discovered or experimentally supported several interesting behaviors: at neutral pH, a pyrimidine-motif triplex can be formed; the parallel arrangement was not only possible but also dominant over the antiparallel arrangement for a purine-motif triplex. We demonstrated that our method is a versatile analytical tool in studying structural aspects of nucleic acids, particularly non-classical DNA structures, and provides insights into physical mechanism of such structures.

Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3′-amino-2′-O,4′-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2′-O and 4′-C of the sugar moiety were bridged with the methylene chain and 3′-O was replaced by 3′-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.

Presence of Divalent Cation Is Not Mandatory for the Formation of Intramolecular Purine-Motif Triplex Containing Human c-jun Protooncogene Target

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.

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.

Interrupted 2′-O,4′-C-Aminomethylene Bridged Nucleic Acid Modification Enhances Pyrimidine Motif Triplex-Forming Ability and Nuclease Resistance Under Physiological Condition

Due to 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 regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′- BNANC) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2′,4′-BNANC modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2′,4′-BNANC modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2′,4′-BNANC modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2′,4′-BNANC-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2′,4′-BNANC-modified positions in TFO. We conclude that the interrupted 2′,4′-BNANC modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.

Promotion of triplex formation by 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification

We examined the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid (3'-amino-2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 3'-amino-2',4'-BNA modified TFO was significantly higher than that observed with unmodified TFO. The 3'-amino-2',4'-BNA modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 3'-amino-2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Thermodynamic and Kinetic Effects of Morpholino Modification on Pyrimidine Motif Triplex Nucleic Acid Formation under Physiological Condition

Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the MOR-modified TFO in the free state relative to the corresponding unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. This information will be valuable for designing novel chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, leading to progress in therapeutic applications of the antigene strategy in vivo.

Promotion of Triplex Formation by 2'-O,4'-C-Aminomethylene Bridged Nucleic Acid (2',4'-BNANC) Modification

We examined the effect of 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNA(NC)) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 2',4'-BNA(NC) modified TFO was significantly higher than that observed with unmodified TFO. The 2',4'-BNA(NC) modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 2',4'-BNA(NC) backbone modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Effect of ENA modification of triplex-forming oligonucleotide on pyrimidine motif triplex formation

We examined the effect of 2'-O,4'-C-ethylene bridged nucleic acid (ENA) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with ENA-modified TFO was 20 times larger than that observed with unmodified TFO. The number and position of the ENA modification introduced into the 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 itself of the ENA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. The present results certainly support the idea that the ENA backbone modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Synergistic stabilization of triplex by combination of comb-type cationic copolymer and 2',4'-BNA

We examined the effect of the combination of the two triplex-stabilizing factors, poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO), on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The combination of both stabilizing factors that was the triplex involving the 2',4'-BNA TFO in the presence of the copolymer synergistically increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the combination of the stabilizing factors can be a key method for triplex stabilization and may lead to progress in therapeutic applications of the antigene strategy in vivo.

Promotion of triplex formation by morpholino modification: thermodynamic and kinetic studies.

We examined the effect of morpholino (MOR) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with MOR-modified TFO was approximately 60 times larger than that observed with unmodified TFO. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the MOR backbone modification resulted mainly from the considerable increase in the association rate constant. The present results certainly support the idea that the MOR backbone modification of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

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.

Triplex formation involving 2',4'-BNA with isoquinolone base analogue: efficient and selective recognition of C:G interruption.

We examined the thermodynamic properties of 2',4'-bridged nucleic acid containing 1-isoquinolone as a nucleobase (QB) to recognize a C interruption in the homopurine strand of the target duplex for pyrimidine motif triplex formation at neutral pH. The triplex formation involving triplex-forming oligonucleotide with QB is highly sequence-selective to specifically recognize C:G target base pair rather than the other G:C, T:A, or A:T base pairs. QB.C:G triad gives significantly larger binding constant than T.C:G triad, which has been known to be the most stable combination in natural base.C:G triad. Our results certainly support the idea that QB could be a key nucleoside to recognize a C interruption in the homopurine strand of the target duplex with high binding affinity and selectivity, and reduce the restriction of target sequences for triplex formation.

Promotion of triplex formation by N3'-->P5' phosphoramidate modification: thermodynamic and kinetic studies.

I examined the effect of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. Both thermodynamic and kinetic analyses have indicated that the PN modification of TFO increased the binding constant of the pyrimidine motif triplex formation at pH 6.8 by nearly 2 orders of magnitude. Kinetic data have also demonstrated that the observed increase in the binding constant at neutral pH by the PN modification resulted mainly from the considerable decrease in the dissociation rate constant rather than the increase in the association rate constant. The present results certainly support the idea that the PN modification of TFO could be a key modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Synergistic stabilization of triplex by combination of comb-type cationic copolymer and oligo-N3'-->P5' phosphoramidates.

In the present study, we describe rapid formation of stable pyrimidine motif triplex at physiological pH. The triplex formation was achieved by the synergistic effect of poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO). Either the PLL-g-Dex copolymer or the PN modification alone increased the binding constant by nearly two orders of magnitude for the triplex formation at neutral pH. The combination of both stabilizing factors that was the triplex formation with the PN TFO in the presence of the copolymer increased the binding constant by nearly four orders of magnitude. The kinetic study indicated that the copolymer increased the association rate constant, whereas the PN modification decreased the dissociation rate constant. No negative interference between these stabilizing effects was observed. The kinetically orchestrated effects in which the copolymer and the PN TFO contribute to distinct ingredients in triplex equilibrium achieved the rapid formation of the stable triplex.

Triplex formation involving 2',4'-BNA with 2-pyridone base analogue: Efficient and selective recognition of C:G interruption

We examined the thermodynamic properties of 2',4'-bridged nucleic acid containing 2-pyridone as a nucleobase (PB) to recognize a C interruption in the homopurine strand of the target duplex for pyrimidine motif triplex formation at neutral pH. The triplex formation involving triplex-forming oligonucleotide with PB is highly sequence-selective to specifically recognize C:G target base pair rather than the other G:C, T:A, or A:T base pairs. PB.C:G triad gives significantly larger binding constant than T.C:G triad, which has been known to be the most stable combination in natural base.C:G triad. Our results certainly support the idea that PB could be a key nucleoside to recognize a C interruption in the homopurine strand of the target duplex with high binding affinity and selectivity, and reduce the restriction of target sequences for triplex formation.

Strand breaks in whole plasmid dna produced by the decay of (125)I in a triplex-forming oligonucleotide.

DNA strand breaks produced by the decay of (125)I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the (125)I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of (125)I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the (125)I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.

Selective recognition of pyrimidine motif triplexes by a protein encoded by the bacterial transposon Tn7

The bacterial transposon Tn7 is distinguished among mobile genetic elements by its targeting abilities. Recently, we reported that Tn7 is able to selectively insert adjacent to triple-helical DNA. The binding of TnsC, a Tn7-encoded protein, to the triplex DNA target leads to the specific transposition of Tn7 adjacent to both inter- and intramolecular pyrimidine motif triplexes. Here, we further probe how Tn7 targets triplex DNA. We report that TnsC discriminates between different types of triplexes, showing binding preference for pyrimidine but not for purine motif intermolecular triplex DNA. The binding preferences of TnsC and the Tn7 insertion profiles were obtained using psoralenated, triplex-forming oligonucleotides annealed to plasmid DNAs. Although the presence of psoralen is not required for targeting nor is it alone able to attract TnsC, we show that the location of psoralen within the pyrimidine motif triplex does alter the position of Tn7 insertion relative to the triplex. Comparison between the triplex-targeting pathway and the highly site-specific targeting pathway mediated by the binding of the Tn7-encoded protein, TnsD, to the unique site attTn7, suggests that similar structural features within each target DNA are recognized by TnsC, leading to site-specific transposition. This work demonstrates that a prokaryotic protein involved in the targeting and regulation of Tn7 translocation, TnsC, can selectively recognize pyrimidine motif triplexes.