Hybrid DNA i-motif: Aminoethylprolyl-PNA (pC5) enhance the stability of DNA (dC5) i-motif structure

This report describes the synthesis of C-rich sequence, cytosine pentamer, of aep-PNA and its biophysical studies for the formation of hybrid DNA:aep-PNAi-motif structure with DNA cytosine pentamer (dC₅) under acidic pH conditions. Herein, the CD/UV/NMR/ESI-Mass studies strongly support the formation of stable hybrid DNA i-motif structure with aep-PNA even near acidic conditions. Hence aep-PNA C-rich sequence cytosine could be considered as potential DNA i-motif stabilizing agents in vivo conditions.     

i-motif solution structure and dynamics of the d(AACCCC) and d(CCCCAA) tetrahymena telomeric repeats

Using NMR methods, we have resolved the i-motif structures formed by d(AACCCC) and by d(CCCCAA), two versions of the DNA sequence repeated in the telomeric regions of the C-rich strand of tetrahymena chromosomes. Both oligonucleotides form fully symmetrical i-motif tetramers built by intercalation of two hemiprotonated duplexes containing four C•C⁺ pairs. The structures are extremely stable. In the tetramer of d(AACCCC), the outermost C•C⁺ pairs are formed by the cytidines of the 5′ ends of the cytidine tracts. A2 forms an A2•A2 (H6trans–N7) pair stacked to C3•C3⁺ and cross-strand stacked to A1. At 0°C, the lifetimes of the hemiprotonated pairs range from 1 ms for the outermost pair to ~1 h for the innermost pairs. The tetramer of d(CCCCAA) adopts two distinct intercalation topologies in slow conformational exchange. One, whose outermost C•C⁺ pairs are built by the cytidines of the 5′ end and the other by those of the 3′ end. In both topologies, the adenosine bases are fairly well stacked to the adjacent C•C⁺ pairs. They are not paired but form symmetrical pseudo-pairs with their H6cis amino proton and N1 nitrogen pointing towards each other.     

Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.     

Calorimetric unfolding of the bimolecular and i-motif complexes of the human telomere complementary strand, d(C(3)TA(2))(4)

A combination of spectroscopic and calorimetric techniques is used to determine the unfolding thermodynamics of the complexes formed by the complementary sequence of the human telomere, d(C(3)TA(2))(4), in the pH range of 4.2 to 6. Calorimetric melting curves show biphasic transitions; both transitions are shifted to higher temperatures as the pH is decreased, indicative of cytosine protonation, which favors the formation of C*C(+) base pairs. Furthermore, the transition temperature, T(M), of the lower transition depends on strand concentration, while the T(M) of the higher transition is independent of strand concentration, indicating the following sequential melting: bimolecular complex(s)-->intramolecular complex-->random coil. The thermodynamic profiles for the formation of each complex, bimolecular and i-motif reveals small favorable free energy terms resulting from favorable enthalpy-unfavorable entropy compensations, uptake of protons, marginal uptake of counterions (i-motif) and marginal release of water molecules (i-motif). Furthermore, an enthalpy of 3.2 kcal/mol (bimolecular complex) and 5.0 kcal/mol (i-motif) is estimated for a single C*C(+)/C*C(+) base-pair stack.     

Molecular dynamics simulation of the DNA triplex d(TC)5.d(GA)5.d(C+T)5.

A molecular dynamics simulation of the DNA triple helix d(TC)5.d(GA)5.d(C+T)5 is described (C+ represents a protonated cytosine residue). The simulation has been performed using the program AMBER 3.1 and includes counterions and explicit solvent under periodic boundary conditions. Both the dynamic and time-averaged behaviour of the system has been analysed. Considerable deviations from the fibre-diffraction model for DNA triple helix structure are observed, including the repuckering of the purine strand sugars that has been identified in some nuclear magnetic resonance (n.m.r.) studies. The simulation suggests that this conformational change may be driven by the possibility of improved interactions between the phosphate groups of this strand and both the solvent and counterions. Several examples of a particular conformational transition are observed, involving correlated changes in the backbone angles alpha and gamma. These transitions provide a possible explanation for some unusual n.m.r. data that have been reported. The structure of the triple helix major groove also suggests an explanation for the observed stabilization of DNA triplexes by polyvalent cations, and their ability to interact with drugs that bind in the minor groove of DNA duplexes.     

Structure, dynamics, and thermodynamics of mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2

The structures and hydrogen exchange properties of the mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2 have been studied by high-resolution nuclear magnetic resonance. Both the adenine-adenine and thymine-thymine mismatches are intercalated in the duplexes. The structures of these self-complementary duplexes are symmetric, with the two strands in equivalent positions. The evidence indicates that these mismatches are not stably hydrogen bonded. The mismatched bases in both duplexes are in the anti conformation. The mismatched thymine nucleotide in d(CCCTGGG)2 is intercalated in the duplex with very little distortion of the bases or sugar-phosphate backbone. In contrast, the bases of the adenine-adenine mismatch in d(CCCAGGG)2 must tilt and push apart to reduce the overlap of the amino groups. The thermodynamic data show that the T-T mismatch is less destabilizing than the A-A mismatch when flanked by C-G base pairs in this sequence, in contrast to their approximately equal stabilities when flanked by A-T base pairs in the sequence d(CAAAXAAAG.CTTTYTTTG) where X and Y = A, C, G, and T [Aboul-ela, F., Koh, D., & Tinoco, I., Jr. (1985) Nucleic Acids Res. 13, 4811]. Although the mechanism cannot be determined conclusively from the limited data obtained, exchange of the imino protons with solvent in these destabilized heteroduplexes appears to occur by a cooperative mechanism in which half the helix dissociates.     

Studies on formation and stability of the d[G(AG)5]* d[G(AG)5]·d[C(TC)5] and d[G(TG)5]* d[G(AG)5]· d[C(TC)5] triple helices

We have targeted the d[G(AG)₅] · d[C(TC)₅] duplex for triplex formation at neutral pH with either d[G(AG)₅] or d[G(TG)₅]. Using a combination of gel electrophoresis, uv and CD spectra, mixing and melting curves, along with DNase I digestion studies, we have investigated the stability of the 2:1 pur*pur · pyr triplex, d[G(AG)₅] * d[G(AG)₅] · d[C(TC)₅], in the presence of MgCl₂. This triplex melts in a monophasic fashion at the same temperature as the underlying duplex. Although the uv spectrum changes little upon binding of the second purine strand, the CD spectrum shows significant changes in the wavelength range 200–230 nm and about a 7 nm shift in the positive band near 270 nm. In contrast, the 1:1:1 pur/pyr*pur · pyr triplex, d[G(TG)₅] * d[G(AG)₅] · d[C(TC)₅], is considerably less stable thermally, melting at a much lower temperature than the underlying duplex, and possesses a CD spectrum that is entirely negative from 200 to 300 nm. Ethidium bromide undergoes a strong fluorescence enhancement upon binding to each of these triplexes, and significantly stabilizes the pur/pyr*pur · pyr triplex. The uv melting and differential scanning calorimetry analysis of the alternating sequence duplex and pur*pur · pyr triplex shows that they are lower in thermodynamic stability than the corresponding 10-mer d(G₃A₄G₃) · d(C₃T₄C₃) duplex and its pur*pur · pyr triplex under identical solution conditions. © 1997 John Wiley & Sons, Inc.     

A novel end-to-end binding of two netropsins to the DNA decamers d(CCCCCIIIII)2, d(CCCBr5CCIIIII)2and d(CBr5CCCCIIIII)2.

Netropsin is bound to the DNA decamer d(CCCCCIIIII)2, the C-4 bromo derivative d(CCCBr5CCIIIII)2and the C-2 bromo derivative d(CBr5CCCCIIIII)2in a novel 2:1 mode. Complexes of the native decamer and the C-4 bromo derivative are isomorphous, space group P1, unit cell dimensions a = 32.56 A (32.66), b = 32.59 A (32.77), c = 37.64 A (37.71), alpha = 86.30 degrees (86.01 degrees), beta = 84.50 degrees (84.37 degrees), gamma = 68.58 degrees (68.90 degrees) with two independent molecules (A and B) in the asymmetric unit (values in parentheses are for the derivative). The C-2 bromo derivative is hexagonal P61, unit cell dimensions a = b = 32.13 A, c = 143.92, gamma = 120 degrees with one molecule in the asymmetric unit. The structures were solved by the molecular replacement method. The novelty of the structures is that there are two netropsins bound end-to-end in the minor groove of each B-DNA decamer which has nearly a complete turn. The netropsins are held by hydrogen bonding interactions to the base atoms and by sandwiching van der Waal's interactions from the sugar-phosphate backbones of the double helix similar to every other drug.DNA complex. Each netropsin molecule spans approximately 5 bp. The netropsins refined with their guanidinium heads facing each other at the center, although an orientational disorder for the netropsins cannot be excluded. The amidinium ends stretch out toward the junctions and bind to the adjacent duplexes in the columns of stacked symmetry-related complexes. Both cationic ends of netropsin are bridged by water molecules in one of the independent molecules (molecule A) of the triclinic structures and also the hexagonal structure to form pseudo-continuous drug.decamer helices.     

Intramolecular i-motif structure at acidic pH for progressive myoclonus epilepsy (EPM1) repeat d(CCCCGCCCCGCG)n.

The most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type is the expansion of a dodecamer repeat, d(CCCCGCCCCGCG)n. We show that the C-rich strand of this repeat (2-3 copies) forms intercalated i-motif structure at acidic pH as judged by CD spectroscopy and anomalous gel electrophoretic mobility. The stability of the structure increases with the increase in the length of the repeat. Transient formation of stable, folded back structure like i-motif could play an important role in the mechanism of expansion of this repeat.     

Molecular-Mechanical studies of the mismatched base analogs of d(CGCGAATTCGCG)2:d(CGTGAATTCGCG)2, d(CGAGAATTCGCG)2, d(CGCGAATTCACG)2, d(CGCGAATTCTCG)2, and d(CGCAGAATTCGCG)·d(CGCGAATTCGCG)

Molecular-mechanical simulations have been carried out on “mismatched base” analogs of the DNA double-helical structure d(CGCGAATTCGCG)₂, in which the base pairs CG at the 3 and 10 positions have been replaced by CA, AG, TC, and TG base pairs, as well as an insertion analog in which an extra adenine has been incorporated into one strand of the above structure between bases 3 and 4. The results of these simulations (calculated relative stabilities, structures, and nmr ring-current shifts) have been compared with calorimetric and nmr data. The calculated relative stabilities of the double-helical parent dodecamer and the various “wobble” base pairs qualitatively correlate with the experimental melting temperatures. The base-pairing structure for the GT wobble pair is in agreement with that previously determined from nmr experiments. For the GA base pair, the structure with both bases anti has a slightly more favorable energy from base pairing and stacking than a structure with non-Watson-Crick H-bonding with adenine syn, in agreement with nmr experiments. The CA wobble base is calculated to favor an adenine 6NH₂ …? cytosine N3 H-bond over cytosine 4NH₂ …? adenine N1, again, in agreement with nmr experiments. There is no definitive experimental data on the TC base pair, but the existence of (somewhat long and weak) H-bonds involving cytosine 4NH₂ …? thymine 4CO and cytosine N3 …? thymine HN3 seems reasonable. We find a structure in which the extra adenine base of the insertion analogs sits “inside” the double helix.     

Structure of the beta-form of poly d(A).poly d(U)

The crystalline beta-form of the sodium salt of poly d(A).poly d(U) trapped in oriented fibers forms a Watson-Crick base-paired, 10(1) double-helix of pitch 3.2 nm. Two molecules are present in a monoclinic unit cell apparently isomorphous with beta-poly d(A).poly d(T). The two chains in each molecule both carry C2'-endo puckered furanose rings but are conformationally not identical. The orientations of the A:U base-pairs relative to the helix-axis are distinctly different from those in classical B-DNA and the overall morphology of the duplex in which they reside resembles that of the alpha-forms of poly (purine).poly (pyrimidine) DNA duplexes previously reported.     

Molecular mechanical studies of base-pair opening in d(CGCGC):d(GCGCG), dG5·dC5, d(TATAT):d(ATATA), and dA5·dT5 in the B and Z forms of DNA

Molecular mechanical energy refinement of double-helical pentanucleotide tetra-phosphates, d(CGCGC):d(GCGCG), dG₅·dC₅, d(TATAT):d(ATATA), and dA₅ ·dT₅ geometries, are presented in order to examine the energy required to open the Nl(purine) …? N3(pyrimidine) distance (base-pair opening) of a Watson-Crick base pair from its normal value of 3 Å to a value of 6 Å. The structural consequences of forcing base-pair opening is sequence dependent. For both dA₅ ·dT₅ and d(TATAT):d(ATATA), forcing the Nl (AdeKN3 (Thy) distance of the central base pair to a value of 6 Å slides the bases perpendicular to the helix axis forming a low-energy non-Watson-Crick base pair having an adenine amine hydrogen …? thymine carbonyl oxygen hydrogen bond. The two GC sequences behave differently from both AT sequences and differently from each other. Forcing the Nl(Gua) …? N3(Cyt) distance to 6 Å leads to unconventional structures in which hydrogen bonds are formed between the separated bases and the bases above or below them. These structures appear to be trapped in true local minima 6–10 kcal/mol higher in energy than the Watson-Crick structures. Preliminary simulations on d(CGCGC):d(GCGCG) in the Z geometry suggest the reason the Z form may be more refractory to proton exchange than the B form, consistent with experimental observations.     

Formation of the G-quadruplex and i-motif structures in retinoblastoma susceptibility genes (Rb)

The formation of G-quadruplex and i-motif structures in the 5′ end of the retinoblastoma (Rb) gene was examined using chemical modifications, circular dichroism (CD) and fluorescence spectroscopy. It was found that substitutions of 8-methylguanine at positions that show syn conformations in antiparallel G-quadruplexes stabilize the structure in the G-rich strand. The complementary C-rich 18mer forms an i-motif structure, as suggested by CD spectroscopy. Based on the C to T mutation experiments, C bases participated in the C–C⁺ base pair of the i-motif structure were determined. Experiments of 2-aminopurine (2-AP) substitution reveal that an increase of fluorescence in the G-quadruplex relative to duplex is attributed to unstacked 2-AP within the loop of G-quadruplex. The fluorescence experiments suggest that formation of the G-quadruplex and i-motif can compete with duplex formation. Furthermore, a polymerase arrest assay indicated that formation the G-quadruplex structure in the Rb gene acts as a barrier in DNA synthesis.     

Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.     

Polarized Raman scattering from oriented single microcrystals of d(A5T5)2 and d(pTpT)

Polarized Raman spectra have been obtained from single microcrystals of the duplex of the decamer d(A₅T₅)₂ using a Raman microscope. This is the first report of Raman spectra from a crystal of a deoxyoligomer that contains only long, nonalternating sequences of adenine and thymine. Sequences containing d(A)_n and d(T)_n are of interest in view of recent suggestions that they induce bends in DNA and that they might exist in a nonstandard B-conformation. Polarized Raman spectra of a crystal of d(pTpT) have also been obtained. Both crystals display Raman bands whose intensities are very sensitive to the orientation of the crystal with respect to the direction of polarization of the incident laser beam. These spectra indicate that the helical axes of the oligonucleotides are parallel to the long axes of the crystals and that the d(A₅T₅)₂ is not appreciably bent in the crystal. The Raman spectrum from the d(pTpT) crystal indicates that all of the furanose ring puckers are in a C2′-endo configuration since only the C2′-endo marker band at 835 ± 5 cm^?1 is present. Crystals of d(A₅T₅)₂ show measurable Raman intensities in both the 838- and 816-cm^?1 bands. This indicates the presence of both the C2′-endo and C3′-endo, or possibly other non-C2′-endo, furanose conformations. The 816-cm^?1 band is weak so that only a small fraction of the residues are estimated to be in the non-C2′-endo conformation. In both the d(pTpT) and d(A₅T₅)₂ crystals the intensity of the bands due to vibrations of the backbone show only a small dependence on orientation of the crystals. This result is explained by the low symmetry of the puckered sugar rings. It is concluded that Raman spectra obtained from oligonucleotide crystals in which the orientation of the crystal axes to the laser polarization is not carefully controlled may contain intensity artifacts that are due to polarization effects.     

Structure of the tubulin dimer

Microtubules are formed from a 110,000-dalton dimeric subunit called tubulin. Two forms of 55,000-dalton monomer, alpha and beta, are found in all microtubule preparations. The dimers could thus theoretically be either heterodimers (alphabeta) or homodimers (alphaalpha and betabeta). This problem was investigated by stigated by chemical cross-linking using several bifunctional reagents, of which one, dimethyl-3,3-(tetrame thylenedioxy) dipropionimidate dihydrochloride (DTDI), was able to make intradimer bonds in tubulin. When soluble chick brain tubulin was cross-linked with DTDI and analyzed by electrophoresis in an acrylamide gel system capable of resolving alphaalpha, alphabeta, and betabeta, 60 to 90% of the cross-linked dimer was alphabeta. If tubulin was incubated at 24 degrees prior to cross-linking with DTDI the total yield of cross-linked dimer increased with time, indicating that tubulin was forming loose aggregates. The relative amounts of cross-linked dimer alphaalpha and betabeta also increase with time, indicating that soluble tubulin is largely alphabeta, and suggesting that cross-linked alphaalpha and betabeta arise from nonspecific aggregation during tubulin purification. The aggregation observed by cross-linking with DTDI was strongly influenced by colchicine and Vinca alkaloids in a pattern similar to the effects of these drugs on tubulin polymerization.     

Re-refinement of the B-dodecamer d(CGCGAATTCGCG) with a comparative analysis of the solvent in it and in the Z-hexamer d(5BrCG5BrCG5BrCG)

The effects of X-ray refinement algorithm on parameters characterising nucleic acid structure are analysed following the re-refinement of the B-dodecamer d(CGCGAATTCGCG). The main conclusions are the following. Mean deviations of main chain torsion angles between the two refinements average 12.6 degrees. Phase angle of pseudorotation for sugar puckers vary between 100 degrees and 180 degrees in the present refinement with amplitude of pucker around 30 degrees. On the other hand, the helical parameters have mean deviations less than 2 degrees. At most half of the assigned solvent positions are within 2 A in both refinements. In the second part of the work, plots of temperature factors (B's) versus occupancies (Q's) for solvent peaks have been analysed in the B-dodecamer and in the Z-hexamer d(5BrCG5BrCG5BrCG). Owing to the poor statistics, some of those conclusions should be regarded as tentative. Occupancy appears to depend on the number of contacts made by the solvent peak with the nucleic acid while temperature factor does not. Except when engaged in particular interaction sites, solvent molecules bound to phosphates have a tendency for high B's and variable Q's. Water molecules bound to polar atoms of the bases occupy various positions in the B-Q diagram. Particularly striking is the behavior of the water molecules belonging to the B-spine and to the Z-spine: the spread in occupancy of water molecules in the hydration spine of the Z-oligomer is larger than in the hydration spine of the B-oligomer. An opposite tendency is observed for the temperature factors. The first observation might reflect the special mobility of the water molecules building up the spine hydration in the Z-form where it continues without interruption from one hexamer to the next. In the B-form, on the contrary, the spine is restricted to the center part of the dodecamer. The second observation might reflect the sharpness of the local attractive potential in the Z-form and its broadness in the B-form. In both cases, dipole reorientations would occur, leading to a high local dielectric constant: in the Z-form, through water molecules hopping from one site to another and, in the B-form, because of rotational freedom.     

Infrared spectral studies of the non regularly alternating purine-pyrimidine hexamers d(m5CGGCM5CG), d(CBr8GGCCBr8G) and d(CGCGGC)

The oligonucleotides d(m5CGGCm5CG), d(CBr8GGCCBr8G) and d(CGCGGC) have been prepared and studied by infrared spectroscopy. The three sequences contain two GC pairs which are out of purine-pyrimidine alternation with the rest of the sequence. From the IR data of the d(m5CGGCm5CG) hexamer, it is shown that all of the dG residues adopt a syn conformation. The marker IR bands for the C3' endo syn conformation are at 1410, 1354, 1320 and 925 cm-1 whereas those for the C2' endo anti conformation at 1420, 1374 and 890 cm-1 are clearly absent. This result implies that the two adjacent guanines of the d(m5CGGCm5CG) sequence are in syn conformation. It is suggested that duplex formation occurs in d(CGCGGC) films and that all of the guanines are in syn conformation. In contrast, the central non-brominated guanine of the d(CBr8GGCCBr8G) hexamer is found in anti conformation, as expected in a Z type structure of the non-alternating region.     

Infrared Spectral Studies of the Non Regularly Alternating Purine-Pyrimidine Hexamers d(m5CGGCM5CG), d(CBr8GGCCBr8G) and d(CGCGGC)

Abstract

The oligonucleotides d(m⁵CGGCm⁵CG), d(CBr⁸GGCCBr⁸G) and d(CGCGGC) have been prepared and studied by infrared spectroscopy. The three sequences contain two GC pairs which are out of purine-pyrimidine alternation with the rest of the sequence. From the IR data of the dlm⁵CGGCm^CG) hexamer, it is shown that all of the dG residues adopt a syn conformation. The marker IR bands for the C3′ endo syn conformation are at 1410, 1354, 1320 and 925 cm^?1 whereas those for the C2′ endo and conformation at 1420, 1374 and 890 cm^?1 are clearly absent. This result implies that the two adjacent guanines of the d(m⁵CGGCm⁵CG) sequence are in syn conformation. It is suggested that duplex formation occurs in d(CGCGGC) films and that all of the guanines are in syn conformation. In contrast, the central non-brominated guanine of the dlCBr⁸GGCCBr⁸G) hexamer is found in ami conformation, as expected in a Z type structure of the non-alternating region.