Biomedical subjects

J Liquier

Publications and source records attributed to J Liquier.

At least 19 recordsLinked to original sources

PubMed2007

Mechanism of copper mediated triple helix formation at neutral pH in Drosophila satellite repeats.

The highly repeated Drosophila melanogaster AAGAGAG satellite sequence is present at each chromosome centromere of the fly. We demonstrate here how, under nearly physiological pH conditions, these sequences can form a pyrimidine triple helix containing T.A-T and CCu.G-C base triplets, stabilized by Cu2+ metal ions in amounts mirroring in vivo concentrations. Ultraviolet experiments were used to monitor the triple helix formation at pH 7.2 in presence of Cu2+ ions. Triplex melting is observed at 23 degrees C. Furthermore, a characteristic signature of triple helix formation was obtained by Fourier transform infrared spectroscopy. The stabilization of the C.G-C base triplets at pH 7.2 is shown to occur via interactions of Cu2+ ions on the third strand cytosine N3 atom and on the guanine N7 atom of the polypurine target strand forming CCu.G-C triplets. Under the same neutral pH conditions in absence of Cu2+ ions, the triple helix fails to form. Possible biological implications are discussed.

Animals↗

PubMed2005

Characterization of parallel and antiparallel G-tetraplex structures by vibrational spectroscopy.

A series of G-rich oligonucleotides able to form tetraplexes has been studied by FTIR spectroscopy. Characteristic markers of the formation of guanine tetrads are given. Moreover, we propose a new marker discriminating between parallel and antiparallel tetraplexes: the position of the C6O6 guanine carbonyl stretching vibration. In intermolecular parallel tetrameric structures formed by four separate strands this absorption is observed at 1693 cm-1 while for antiparallel tetrameric structures, either intramolecular or formed by dimerization of hairpins, this vibrational mode is observed at 1682 cm-1. These shifts to higher wavenumbers, when compared to the position of a free guanine C6O6 carbonyl stretching vibration observed at 1666 cm-1(Deltanu=27 cm-1 for parallel tetraplexes and Deltanu=16 cm-1 for antiparallel tetraplexes) reflect different strand orientations in the structures. This marker has been used to evidence the possibility of an antiparallel-parallel tetraplex reorganization for Oxytricha nova d(G4T4G4) and d((G4T4)3G4) and human d(G3T2AG3) telomeric sequences induced by Na+/K+ or Na+/Ca2+ ion exchange. Formation of the guanine tetrads, characterization of the phosphate geometries and of the sugar conformations have also been obtained by FTIR for the different tetraplexes.

Animals↗

PubMed2005

Parallel DNA double helices incorporating isoG or m5isoC bases studied by FTIR, CD and molecular modeling.

FTIR spectroscopy has been used to follow the formation of parallel stranded DNA duplexes incorporating isoG or m5isoC bases and determine their base pairing scheme. The results are discussed in comparison with data concerning anti-parallel duplexes with comparable base composition and sequence. In duplexes containing A-T and isoG-C or m5isoC-G base pairs shifts of the thymine C2=O2 and C4=O4 carbonyl stretching vibrations (to lower and higher wavenumbers, respectively, when compared to their positions in classical cis Watson-Crick (WC) base pairs) reflect the formation of trans Watson-Crick A-T base pairs. All carbonyl groups of cytosines, m5isocytosines, guanines and isoguanines are found to be involved in hydrogen bonds, indicative of the formation of isoG-C and m5isoC-G base pairs with three hydrogen bonds. Molecular modeling shows that both structures form regular right handed helices with C2'endo sugar puckers. The role of the water content on the helical conformation of the parallel duplexes has been studied by FTIR and CD. It is found that a conformational transition similar to the B --> A transition observed for anti-parallel duplexes induced by a decrease of the water content of the samples can occur for these parallel duplexes. Their helical flexibility has been evidenced by FTIR studies on hydrated films by the emergence of absorption bands characteristic of A type geometry, in particular by an S-type --> N-type repuckering of the deoxyribose. All sugars in the parallel duplex with alternating d(isoG-A)/d(C-T) sequence can adopt an N-type geometry in low water content conditions. The conformational transition of the parallel hairpin duplex with alternating d(isoG-A)/d(C-T) sequence was followed by circular dichroism in water/trifluoroethanol solutions and its free energy at 0 degrees C was estimated to be 6.6 +/- 0.3 kcal mol(-1).

Base Composition↗

PubMed2005

Hydration and conformational transitions in DNA, RNA, and mixed DNA-RNA triplexes studied by gravimetry and FTIR spectroscopy.

We have studied by gravimetric measurements and FTIR spectroscopy the hydration of duplexes and triplexes formed by combinations of dA(n), dT(n), rA(n), and rU(n) strands. Results obtained on hydrated films show important differences in their hydration and in the structural transitions which can be induced by varying the water content of the samples. The number of water molecules per nucleotide (w/n) measured at high relative humidity (98% R.H.) is found to be 21 for dA(n).dT(n) and 15 for rA(n).rU(n). Addition of a third rU(n) strand does not change the number of water molecules per nucleotide: w/n=21 for rU(n)*dA(n).dT(n) and w/n=15 for rU(n)*rA(n).rU(n). On the contrary, the addition of a third dT(n) strand changes the water content but in a different way, depending whether the duplex is DNA or RNA. Thus, a loss of four water molecules per nucleotide is measured for dT(n)*dA(n).dT(n) while an increase of two water molecules per nucleotide is observed for dT(n)*rA(n).rU(n). The final hydration is the same for both triplexes (w/n=17). The desorption profiles obtained by gravimetry and FTIR spectroscopy are similar for the rA(n).rU(n) duplex and the rU(n)*rA(n).rU(n) triplex. On the contrary, the desorption profiles of the dA(n).dT(n) duplex and the triplexes formed with it (rU(n)*dA(n).dT(n) and dT(n)*dA(n).dT(n)) are different from each other. This is correlated with conformational transitions induced by varying the hydration content of the different structures, as shown by FTIR spectroscopy. Modifications of the phosphate group hydration and of the sugar conformation (S to N type repuckering) induced by decrease of the water content are observed in the case of triplexes formed on the dA(n).dT(n) duplex.

DNA↗

PubMed2004

Tetraplex structure formation in the thrombin-binding DNA aptamer by metal cations measured by vibrational spectroscopy.

Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations.

Aptamers, Nucleotide↗

PubMed2001

Parallel and antiparallel GG.C base triplets in purpur.pyr triple helices formed with (GA) third strands.

Triple helices with G*G.C and A*A.T base triplets with third GA strands either parallel or antiparallel with respect to the homologous duplex strand have been formed in presence of Na (+) or Mg(2+) counterions. Antiparallel triplexes are more stable and can be obtained even in presence of only monovalent Na(+) counterions. A biphasic melting has been observed, reflecting third strand separation around 20 degrees C followed by the duplex -> coil transition around 63 degrees C. Parallel triplexes are far less stable than the antiparallel ones. Their formation requires divalent ions and is observed at low temperature and in high concentration conditions. Different FTIR signatures of G*G.C triplets in parallel and antiparallel triple helices with GA rich third strands have been obtained allowing the identification of such base triplets in triplexes formed by nucleic acids with heterogeneous compositions. Only S-type sugars are found in the antiparallel triplex while some N-type sugar conformation is detected in the parallel triplex.

Adenine↗

PubMed2000

Parallel self-associated structures formed by T,C-rich sequences at acidic pH.

Oligonucleotides of nonregular heteropyrimidine sequences incorporating or not incorporating purine residues 5'-d(ACTCCCTTCTCCTCTCTA), 5'-d(ACTCCCTGGTCCTCTCTA), 5'-d(TCTCTCCTGGTCCCTCC), and 5'-d(TCTCTCCTCTTCCCTCC) can form self-associated parallel-stranded (ps) structures at pH 4-5.5. The ps structures were identified by studying at neutral and acidic pH UV melting transitions, FTIR spectra, and fluorescence of pyrene-labeled oligonucleotides as well as by chemical joining of 5'-phosphorylated oligonucleotides. A gel electrophoresis run for oligonucleotides 5'-d(TCTCTCCTCTTCCCTCC) and 5'-d(ACTCCCTTCTCCTCTCTA) has shown the formation of homoduplexes at low DNA strand concentrations. Ps structures are held by C-C(+) base pairs and have N- and S-types of sugar puckering as detected by FTIR spectroscopy in the millimolar concentration range. Guanine inserts as well as thymine and purine inserts into an oligomeric cytosine sequence make the formation of the tetraplex i-motif unfavorable. MvaI restriction endonuclease, which recognizes the CCT/AGG sequence in DNA, does not cleave parallel pseudosubstrates.

Acids↗

PubMed2000

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn(2+) ions.

We present evidence of formation of an intramolecular parallel triple helix with T*A.T and G*G.C base triplets (where * represents the hydrogen bonding interaction between the third strand and the duplex while. represents the Watson-Crick interactions which stabilize the duplex). The third GT strand, containing seven GpT/TpG steps, targets the polypurine sequence 5'-AGG-AGG-GAG-GAG-3'. The triple helix is obtained by the folding back twice of a 36mer, formed by three dodecamers tethered by hydroxyalkyl linkers (-L-). Due to the design of the oligonucleotide, the third strand orientation is parallel with respect to the polypurine strand. Triple helical formation has been studied in concentration conditions in which native gel electrophoresis experiments showed the absence of intermolecular structures. Circular dichroism (CD) and UV spectroscopy have been used to evidence the triplex structure. A CD spectrum characteristic of triple helical formation as well as biphasic UV and CD melting curves have been obtained in high ionic strength NaCl solutions in the presence of Zn(2+) ions. Specific interactions with Zn(2+) ions in low water activity conditions are necessary to stabilize the parallel triplex.

Base Pairing↗

PubMed1998

FTIR and UV spectroscopy of parallel-stranded DNAs with mixed AT/GC sequences and their AT/IC analogues.

The infrared spectra of parallel-stranded (ps) hairpin duplexes with mixed A*T/G*C composition and either isolated or sequential G*C pairs were studied in comparison with antiparallel-stranded (aps) duplexes and a corresponding set of molecules with hypoxanthine as a G base analogue lacking the exocyclic amino group. The ps duplexes showed the characteristic bands for the C2=O2 and C4=O4 stretching vibrations of thymine residues in trans-Watson-Crick A*T pairing at 1683 and 1668 cm-1. The latter band was superimposed on the stretching vibration of the free C6=O6 group of guanine. Substitution of guanine by hypoxanthine inhibited the formation of ps hairpin duplexes whatever the sequence, demonstrating that in the H-bonding between G and C the 2-NH2 group is necessary for stabilizing all of the investigated ps duplexes. This result is in agreement with a model of trans-Watson-Crick G*C base pairs with two H-bonds [N2H2(G)-N3(C) and N1H(G)-O2(C)]. However, trans-Watson-Crick A*T and G*C base pairs with two H-bonds are not isomorphous, which may explain the decreased stability of the ps, but not the aps, duplexes upon increasing the number of A*T/G*C steps. Molecular modeling studies performed on two of the ps duplexes reveal the existence of propeller twist for avoiding a clash between the N2(G) and N4(C) amino groups, and favorable stacking of sequential G*C base pairs. The optimized hairpin ps duplexes invariably incorporated G*C base pairs with two H-bonds, regardless of the initial structures adopted for the force field calculations.

Adenine↗

PubMed1998

Conformational study of DNA-RNA duplexes containing MMI substituted phosphodiester linkages by FTIR spectroscopy.

Six methylene(methylimino) (MMI, Bhat et al. J. Org. Chem., 61, 8186, 1996) linked oligonucleotides a-f (* = MMI linkage; 5'-GCGT*TT*TT*TT*TT*TGCG-3') containing various combinations of 2'-O-methyl and 2'-fluoro substituent were synthesized as a model to study the global conformational change upon hybridization to the complement RNA. Fourier transform infrared (FTIR) spectroscopic technique has been used to study and compare the influence of these modifications on the solution conformation of 2'-modified MMI DNA-RNA duplexes. FTIR analysis of the single-stranded RNA (5'-CGCAAAAAAAAAACGC-3') and the modified oligonucleotides a-f showed that all sugar residues adopted a C3'-endo conformation (North-type). Stable duplexes were formed when oligonucleotides a-f were hybridized to the complement RNA. These duplexes retained the original C3'-endo conformation for all sugar residues, hallmark of an A-form of duplex. We postulate that the observed preorganization of the sugar residues and oligonucleotides containing 2'-modified MMI modifications may play an important role in both improving the recognition of RNA target and enhancing the stability of duplex formation with RNA.

Carbohydrates↗

PubMed1997

Hydration of the dTn.dAn x dTn parallel triple helix: a Fourier transform infrared and gravimetric study correlated with molecular dynamics simulations.

We present a comparative analysis of the water organization around the dTn.dAn x dTn triple helix and the Watson-Crick double helix dTn.dAn respectively by means of gravimetric measurements, infrared spectroscopy and molecular dynamics simulations. The hydration per nucleotide determined by gravimetric and spectroscopic methods correlated with the molecular dynamics simulations shows that at high relative humidity (98% RH) the triple helix is less solvated than the duplex (17 +/- 2 water molecules per nucleotide instead of 21 +/-1). The experimental desorption curves are different for both structures and indicate that below 81% RH the triplex becomes more hydrated than the duplex. At this RH the FTIR spectra show the emergence of N-type sugars in the adenosine strand of the triplex. When the third strand is bound in the major groove of the Watson-Crick duplex molecular dynamics simulations show the formation of a spine of water molecules between the two thymidine strands.

DNA↗

PubMed1997

A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3'--> P5' phosphoramidate linkages.

Non-denaturing gel retardation assay, DNA melting experiments and FTIR spectroscopy were used to characterize the triple helix formed by a 15mer 2'-deoxythymidylate with N3'-->P5'phosphoramidate linkages with its target sequence. The results indicate that: (i) the pentadecadeoxythymidylate with phosphoramidate linkages [dT15(np)] is highly potent to form a triple helix with a dT15*dA15target duplex through Hoogsteenbase-pairing; (ii) it forms a dT15(np)*dA15xdT15(np) triplex with the single-stranded oligo-2'-deoxyadenylate (dA15) without detectable double-helical intermediate; (iii) it does not only form a triple helix on the dT15*dA15target duplex, but also partially displaces the dT15 strand from the dT15*dA15duplex to form a dT15(np)*dA15xdT15(np) complex.

DNA↗

PubMed1996

Parallel and antiparallel A*A-T intramolecular triple helices.

Intramolecular triple helices have been obtained by folding back twice oligonucleotides formed by decamers bound by non-nucleotide linkers: dA10-linker-dA10-linker-dT10 and dA10-linker-dT10-linker-dA10. We have thus prepared two triple helices with forced third strand orientation, respectively antiparallel (apA*A-T) and parallel (pA*A-T) with respect to the adenosine strand of the Watson-Crick duplex. The existence of the triple helices has been shown by FTIR, UV and fluorescence spectroscopies. Similar melting temperatures have been obtained in very different oligomer concentration conditions (micromolar solutions for thermal denaturation classically followed by UV spectroscopy, milimolar solutions in the case of melting monitored by FTIR spectroscopy) showing that the triple helices are intramolecular. The stability of the parallel triplex is found to be slightly lower than that of the antiparallel (deltaT(m) = 6 degrees C). The sugar conformations determined by FTIR are different for both triplexes. Only South-type sugars are found in the antiparallel triplex whereas both South- and North-type sugars are detected in the parallel triplex. In this case, thymidine sugars have a South-type geometry, and the adenosine strand of the Watson-Crick duplex has North-type sugars. For the antiparallel triplex the experimental results and molecular modeling data are consistent with a reverse-Hoogsteen like third-strand base pairing and South-type sugar conformation. An energetically optimized model of the parallel A*A-T triple helix with a non-uniform distribution of sugar conformations is discussed.

Carbohydrate Conformation↗

PubMed1996

Ligand-induced formation of hoogsteen-paired parallel DNA.

INTRODUCTION: Based on molecular modeling studies, a model has been proposed for intercalation of triple-helix-specific ligands (benzopyridoindole (BPI) derivatives) into triple helices, in which the intercalating compounds interact mainly with the Hoogsteen-paired strands of the triple helix. We set out to test this model experimentally using DNA duplexes capable of forming parallel Hoogsteen base-paired structures. RESULTS: We have investigated the possible formation of a parallel DNA structure involving Hoogsteen hydrogen bonds by thermal denaturation, FTIR spectroscopy and gel-shift experiments. We show that BPI derivatives bind to Hoogsteen base-paired duplexes and stabilize them. The compounds induce a reorganization from a non-perfectly matched antiparallel Watson- Crick duplex into a perfectly matched parallel Hoogsteen-paired duplex. CONCLUSIONS: These results suggest that preferential intercalation of BPI derivatives in triple helices is due to their ability to interact specifically with the Hoogsteen-paired bases. The results are consistent with a model proposed on the basis of molecular modeling studies using energy minimization, and they open a new field of investigations regarding the biological relevance of Hoogsteen base-pairing.

DNA↗

PubMed1995

Sugar conformations in DNA and RNA-DNA triple helices determined by FTIR spectroscopy: role of backbone composition.

We have studied the effect of the nature of the third-strand sugar (ribose or deoxyribose) on the geometry and stability of triple helices with a pyrimidine motif targeting the polypurine tract of the Friend murine retrovirus. Comparison between triplexes containing a third strand formed by a deoxy 13mer d(TCT5C6), the same oligomer but with C5-methylated cytosines d(T5meCT5(5me)C6), and an analogous modified 13mer RNA 2'Omer(UCU5C6) shows that the sugar conformations of the different triple helices, determined by FTIR spectroscopy, differ depending on nature of the third-strand sugar. Pyrimidine*purine-pyrimidine triple-helix formation with the third-strand RNA and the duplex as DNA appears to be associated with a conversion of the duplex part from a B-form secondary structure with S-type sugars to a geometry in which the polypurine strand sugars adopt an N-type conformation. Thermal dissociation of the triplexes was studied by UV absorbance spectroscopy. The most stable triple helix is obtained when the third strand contains 2'-O-methylated ribose sugars.

Base Sequence↗

PubMed1995

FTIR study of a nonclassical dT10*dA10-dT10 intramolecular triple helix.

Many early investigations on triple helices have been devoted to the study of the triplex formed by dT*dA-dT base triplets in which the third strand is oriented parallel to the dA strand. We now describe an intramolecular triple helix with dT*dA-dT base triplets in which the pyrimidine third strand is oriented antiparallel, formed by folding back twice the tridecamer dT10-linker-dA10-linker-dT10 (linker = pO(CH2CH2O)3p). Third-strand base pairing to the target strand, sugar conformation, and thermal denaturation of the triplex have been studied by Fourier transform infrared spectroscopy. Our results confirm than when the third-strand orientation is reversed from parallel to antiparallel with respect to the target strand, the third-strand hydrogen-bonding scheme is changed from Hoogsteen to reverse Hoogsteen. The sugar conformation in this triple helix is of the S type (C2'endo/anti, B family form) from all strands. Our results are discussed with respect to models for triplexes proposed as intermediates in homologous recombination [Zhurkin, V.B., Raghunathan, G., Ulyanov, N.B., Camerini-Otero, R.D., & Jernigan, R.L. (1994) J. Mol. Biol. 239, 181-200].

Animals↗

PubMed1995

Structural characterization of an intramolecular RNA triple helix by NMR spectroscopy.

A chemically synthesized 29-base RNA oligomer, designed to fold to form an intramolecular triple helix at acid pH, has been studied by NMR spectroscopy. The molecule consisted of seven U.A.U or C+.G.C base triples joined by two pyrimidine tetra-loops. The fold was such that the third strand was Hoogsteen base-paired in the major groove of a Watson-Crick paired double helix. The nature and size of the molecule required the use of an assignment strategy using two- and three-dimensional homonuclear methods, complemented by a natural abundance 13C correlation experiment. The assignment of the majority of the exchangeable and non-exchangeable resonances is presented. The data suggest a C3'-endo sugar puckering for all the nucleotides involved in base triples. A preliminary structural model consistent with the NMR data is presented.

Magnetic Resonance Spectroscopy↗

PubMed1995

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.

Base Sequence↗