Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich's ataxia

The onset and progress of Friedreich's ataxia (FRDA) is associated with the genetic instability of the (GAA).(TTC) trinucleotide repeats located within the frataxin gene. The instability of these repeats may involve the formation of an alternative DNA structure. Poly-purine (R)/poly-pyrimidine (Y) sequences typically form triplex DNA structures which may contribute to genetic instability. Conventional wisdom suggested that triplex structures formed by these poly-purine (R)/poly-pyrimidine (Y) sequences may contribute to their genetic instability. Here, we report the characterization of the single-stranded GAA and TTC sequences and their mixtures using NMR, UV-melting, and gel electrophoresis, as well as chemical and enzymatic probing methods. We show that the FRDA GAA/TTC, repeats are capable of forming various alternative structures. The most intriguing is the observation of a parallel (GAA).(TTC) duplex in equilibrium with the antiparallel Watson-Crick (GAA).(TTC) duplex. We also show that the GAA strands form self-assembled structures, whereas the TTC strands are essentially unstructured. Finally, we demonstrate that the FRDA repeats form only the YRY triplex (but not the RRY triplex) at neutral pH and the complete formation of the YRY triplex requires the ratio of GAA to TTC strand larger than 1:2. The structural features presented here and in other studies distinguish the FRDA (GAA)?(TTC) repeats from the fragile X (CGG).CCG), myotonic dystrophy (CTG).(CAG) and the Huntington (CAG).(CTG) repeats.     

5-(1-propargylamino)-2'-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent triplex A6-L-T6-L-T6at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by approximately 12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds approximately 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.     

Sequence-dependent stability of intramolecular DNA triple helices

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

Sequence-Dependent Stability of Intramolecular DNA Triple Helices

Abstract

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

Intramolecular DNA triplexes in supercoiled plasmids. I. Effect of loop size on formation and stability

The capacity of four oligopurine.oligopyrimidine (pur.pyr) sequences with different lengths of interruptions in the center [GAA)4(N)n(GAA)4G) (n = 3, 5, 7, and 9) to adopt intramolecular DNA triplexes was evaluated in recombinant plasmids. The hyperreactive patterns of the pur.pyr inserts to specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) at the base pair level demonstrate that intramolecular triplexes with identical 12-base triads in the stem but with different loop sizes (4, 6, 8, and 10 bases) can form in supercoiled plasmids. Furthermore, the extent of OsO4 modification was measured as a function of temperature and of average negative supercoil density. In addition, the transition free energy of B-DNA to triplexes at pH 4.5 was determined by two-dimensional electrophoresis. These comparative studies show that longer loops require more supercoil energy for triplex formation and are less thermostable than triplexes with shorter loops. Also, it may be that not only the loop size but the base composition of the loop region affects the structural transition and triplex stability. Thus, these results significantly broaden the range of natural pur.pyr sequences that may adopt triplexes.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.     

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

FTIR and UV spectroscopy studies of triplex formation between alpha-oligonucleotides with non-ionic phoshoramidate linkages and DNA targets

The triplexes formed by pyrimidine alpha-oligodeoxynucleotides, 15mers alpha dT(15) or 12mers alpha dCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non-ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester beta-oligodeoxynucleotides (beta-ODNs). Among the alpha-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (alpha dCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the alpha dCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All alpha dCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30 degrees C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65 degrees C). While unfavorable steric effects are overcome with alpha anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate alpha-thymidilate strands (alpha dT(15)PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to beta dA(15) either of alpha dT(15)PN as duplex strand and beta dT(15) as third strand, or of two hydrophobic alpha dT(15)PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the alpha dT(15)PN binds as Hoogsteen third strand.     

Evidence for a DNA triplex in a recombination-like motif: I. Recognition of Watson-Crick base pairs by natural bases in a high-stability triplex

Data are presented on a triplex type with two parallel homologous strands for which triplex formation is almost as strong as duplex formation at least for some sequences and even at pH 7 and 0.2 M NaCl. The evidence mainly rests upon comparing thermodynamic properties of similar systems. A paperclip oligonucleotide d(A12C4T12C4A12) with two linkers C4 obviously can form a triplex with parallel back-folded adenine strand regions, because the single melting transition of this complex splits in two transitions by introducing mismatches only in the third strand region. Respectively, a hairpin duplex d(A12C4T12) and a single strand d(A12) form a triplex as a 1:1 complex in which the second adenine strand is parallel oriented to the homologous one in the Watson-Crick paired duplex. In this system the melting temperature T(m) of the triplex is practically the same as that of the duplex d(A12)-d(T12), at least within a complex concentration range of 0.2-4.0 microM. The melting behaviour of complexes between triplex stabilizing ligand BePI and the system hairpin duplex plus single strand supports the triplex model. Non-denaturing gel electrophoresis suggests the existence of a triplex for a system in which five of the twelve A-T*A base triads are substituted by C-G*C base triads. The recognition between any substituted Watson-Crick base pair (X-Y) in the hairpin duplex d(A4XA7C4T7YT4) and the correspondingly replaced base (Z) in the third strand d(A4ZA7) is mutually selective. All triplexes with matching base substitutions (Z = X) have nearly the same stability (T(m) values from 29 to 33.5 degrees C), whereas triplexes with non-matching substitutions (Z not equal X) show a clearly reduced stability (T(m) values from 15 to 22 degrees C) at 2microM equimolar oligonucleotide concentration. Most nucleic acid triple helices hitherto known are limited to homopurine-homopyrimidine sequences in the target duplex. A stable triplex formation is demonstrated for inhomogeneous sequences tolerating at least 50% pyrimidine content in the homologous strands. On the basis of the surprisingly similar thermodynamic parameters for duplex and triplex, and of the fact that this triplex type seems to be more stable than many other natural DNA triplexes known, and on the basis of semiempirical and molecule mechanical calculations, we postulate bridging interactions of the third strand with the two other strands in the triplex according to the recombination motif. This triplex, denoted by us 'recombination-like form', tolerates heterogeneous base sequences.     

Triplex formation by a psoralen-conjugated oligodeoxyribonucleotide containing the base analog 8-oxo-adenine.

Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.     

Remarkable stabilization of antiparallel DNA triplexes by strong stacking effects of consecutively modified nucleobases containing thiocarbonyl groups

The consecutive arrangement of 2′-deoxy-6-thioguanosines (s⁶Gs) and 4-thiothymidines (s⁴Ts) in antiparallel triplex-forming oligonucleotides (TFOs) considerably stabilized the resulting antiparallel triplexes with high base recognition ability by the strong stacking effects of thiocarbonyl groups. This result was remarkable because chemical modifications of the sugar moieties and nucleobases of antiparallel TFOs generally destabilize triplex structures. Moreover, in comparison with unmodified TFOs, it was found that TFOs containing s⁶Gs and s⁴Ts could selectively bind to the complementary DNA duplex but not to mismatched DNA duplexes or single-stranded RNA.     

Spectroscopic studies on ethidium bromide binding to intramolecular parallel and antiparallel triple helices containing T*A:T and G*G:C triplets

The interaction of ethidium bromide (EB), a DNA intercalator, with two intramolecular triplexes 5'd(G4A4G4-[T4]-C4T4C4-[T4]-G4T4G4), 5'd(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4] represents a stretch of 4 thymine residues) and their precursor duplexes has been investigated by circular dichroism, fluorescence and UV absorption spectroscopy. Binding of EB induces a circular dichroism band in the region around 310 nm which is positive for the duplex forms but negative for the triplex forms. We observed that the binding of EB to the duplex form does not induce the formation of the triplex structures. Thermal denaturation experiments demonstrate that EB stabilizes more the parallel triple helix than the antiparallel one. Analysis of the binding process from fluorescence measurements shows that binding constants to the triple helical forms and to the hairpin reference duplex [T4]-G4A4G4-[T4]-C4T4C4) are close. However the binding site size is larger for the triplexes (4-6 base triplets) than for the duplex (2 base pairs).     

Effect of intermolecular triplex formation on the yield of cyclobutane photodimers in DNA.

We have studied the effect of intermolecular triplexes formation on the yield of cyclobutane photodimers in DNA. DNA duplex within the pyrimidine-purine-pyrimidine triplex d(TC)nd(GA)nd(CT)n is protected from the formation of cyclobutane photodimers in the case of the stabilization of this triplex by acid pH, and in the case of supplementary stabilization by Mg2+ or Zn2+. We have studied pH-independent pyrimidine-purine-purine triplexes stabilized by bivalent cations. In such triplexes, the protection from the formation of [6-4] photodimers is observed, whereas the protection from cyclobutane dimer formation does not take place. The formation of the d(TC)nd(GA)nd(GA)n triplex leads to an inversion of the intensities of cyclobutane CT and TC photodimers. We observed a sharp decrease in photoreactivity with respect to cyclobutane dimers in the duplex tract d(C)18d(G)18 in the presence of Ba2+, Cd2+, Co2+, Mn2+, Zn2+ and Ni2+. The formation of the d(C)nd(G)nd(G)n triplex leads to 'antifootprinting', i.e. an increase in the yield of cyclobutane photodimers.