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RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA.

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.

Bacteriophages↗

PubMed1999

Cisplatin (cis-Pt(NH(3))(2)Cl(2)) and cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) Intrastrand Cross-Linking Reactions at the Telomere GGGT DNA Sequence Embedded in a Duplex, a Hairpin, and a Bulged Duplex: Use of Mg(2+) and Zn(2+) to Convert a Hairpin to a Bulged Duplex.

In the past, we showed that metal species have a high affinity for the central G in the GGG sequence of the duplex d(A(1)T(2)G(3)G(4)G(5)T(6)A(7)C(8)C(9)C(10)A(11)T(12))(2) (G3-D) and that cisplatin (cis-Pt(NH(3))(2)Cl(2)) and G3-D formed an N7-Pt-N7 G(4),G(5) intrastrand cross-link preferentially over the G(3),G(4) adduct ( approximately 25:1). Thus, a putative G(4) monoadduct was postulated to cross-link in the 3'- rather than the normally more favorable 5'-direction. To evaluate this hypothesis and also to explore why the G3-D G(4),G(5) adduct had an unusual hairpin structure, we have now introduced the use of N,N'-dimethylthiourea (DMTU) as a monoadduct trap and have extended the study to a G3-D analogue with a hairpin form, d(A(1)T(2)G(3)G(4)G(5)T(6)T(7)C(8)C(9)C(10)A(11)T(12)) (G3-H). Chemical shift and 2D (1)H and (13)C NMR data indicated that the G3-H hairpin has a stem region with B-form structure and a nonhelical loop region. Zn(2+) or Mg(2+) ions transformed G3-H into a bulged duplex. Downfield shifts of G(4)H8 and G(4)C8 NMR signals indicated that Zn(2+) binds preferentially to G(4)N7. Reaction of cisplatin or cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) with the bulged duplex and hairpin forms of G3-H gave a similar intrastrand cross-link ratio, G(4),G(5):G(3),G(4) = 7:3. This ratio is insensitive to DNA form or Pt leaving group. For G3-D this ratio is lower in the cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) reaction ( approximately 1:1) than in the cisplatin reaction (25:1), indicating that the leaving group influences the cross-linking step for G3-D. The G(4) monoadducts of the cis-Pt(NH(3))(2)Cl(2)-G3-H and -G3-D and the cis-[Pt(NH(3))(2)(H(2)O)(2)](2+)-G3-D reactions were trapped with DMTU, but no monoadduct was trapped in the cis-[Pt(NH(3))(2)(H(2)O)(2)](2+)-G3-H reaction. The results suggest that the respective monoadducts are more long-lived for G3-D. We postulate that the G(5) in the G3-D Cl-G(4) monoadduct is placed in a favorable position to form the cross-link because of a prior conformational change induced by G(4)-A(7) stacking. This accounts for the very high selectivity for 3'-cross-linking. Nevertheless, in all other cases, regardless of the form or conformation, 3'-direction cross-linking is unusually favored at GGGT sequences, suggesting that the sequence itself contributes greatly to the 3'-cross-linking preference; since telomeres have multiple repeats of this GGGT sequence, this finding may have biological relevance.

Journal Article↗

PubMed1999

Crystal structures of two plasmid copy control related RNA duplexes: An 18 base pair duplex at 1.20 A resolution and a 19 base pair duplex at 1.55 A resolution.

The structures of two RNA duplexes, whose sequences correspond to portions of the ColE1 plasmid copy control RNA I and RNA II, have been determined. Crystals containing the 18mers 5'-CA CCGUUGGUAGCGGUGC-3' and 5'-CACCGCUACCAACGGUGC-3' diffract to 1.20 A resolution while those containing the 19mers 5'-GCACCGUUGGUAGCGGUGC-3' and 5'-GCACCGCUACCAACGGUGC-3' diffract to 1.55 A resolution. Both duplexes are standard A form, with Watson-Crick base pairing throughout. Use of anisotropic atomic displacement factors in refinement of the 1.20 A structure dramatically improved refinement statistics, resulting in a final R(free) of 15.0% and a crystallographic R-factor of 11.6%. Perhaps surprisingly, these crystals of the 18 base pair RNA exhibit a 36-fold static disorder, resulting in a structure with a single sugar-phosphate backbone conformation and an averaged base composition at each residue. Since the sugar-phosphate backbone structure is identical in the 36 different nucleotides that are superimposed, there can be no sequence-dependent variation in the structure. The average ribose pucker amplitude is 45.8 degrees for the 18 base pair structure and 46.4 degrees for the 19 base pair structure; these values are respectively 19% and 20% larger than the average pucker amplitude reported from nucleoside crystal structures. A standard RNA water structure, based on analysis of the hydration of these crystal structures and that of the TAR RNA stem [Ippolito, J. A., and Steitz, T. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9819-9824], has been derived, which has allowed us to predict water positions in lower resolution RNA crystal structures. We report a new RNA packing motif, in which three pro-S(p) phosphate oxygens interact with an ammonium ion.

Crystallography, X-Ray↗

PubMed2004

Comparative study of duplex-guided foam sclerotherapy and duplex-guided liquid sclerotherapy for the treatment of superficial venous insufficiency.

PURPOSE: To compare the preliminary results of hemodynamic changes between duplex-guided foam sclerotherapy and duplex-guided liquid sclerotherapy. METHODS: Seventy Seven limbs in 77 patients with isolated greater saphenous vein incompetence were treated with duplex-guided sclerotherapy. Thirty Seven limbs were treated with duplex-guided foam sclerotherapy and the remaining 40 limbs were treated with duplex-guided liquid sclerotherapy. Pretreatment exam was performed using a color duplex scanner and air plethysmography. The sclerosing foam was produced by Tessari's method using 1% and 3% polidocanol. The varicose vein was injected with 2 mL of 1% polidocanol or 1% polidocanol foam, and then 1 mL of 3% polidocanol or 3% polidocanol foam was injected into the greater saphenous vein under duplex guidance. Venous obstruction and recanalization were screened by serial posttreatment duplex examination, and posttreatment air plethysmography analysis was performed 3, 6, 9, and 12 months after the sclerotherapy. RESULTS: Duplex scanning demonstrated complete occlusion in the greater saphenous vein for duplex-guided foam sclerotherapy in 25 limbs (67.6%), which was a significantly higher proportion than for the duplex-guided liquid sclerotherapy (7 limbs, 17.5%, p<0.0001). Recurrent varicose veins were found in 3 patient (8.1%) in the duplex-guided foam sclerotherapy group and 10 (25%) in the duplex-guided liquid sclerotherapy group at 1-year (p=0.048). In duplex-guided foam sclerotherapy, venous filling index values remained normal during the subsequent follow-up examinations, whereas in duplex-guided liquid sclerotherapy, venous filling index began to increase, and there was a significant difference at 6 months between duplex-guided foam sclerotherapy and the duplex-guided liquid sclerotherapy (p<0.0005). At 9 months, there was a significant difference in the residual venous fraction between the two groups, and the residual venous fraction value continued to improve in duplex-guided foam sclerotherapy (p=0.033). CONCLUSIONS: Duplex-guided foam sclerotherapy could have greater promise compared to duplex-guided liquid sclerotherapy in the treatment of superficial venous insufficiency.

Adult↗

PubMed1995

Berenil [1,3-bis(4'-amidinophenyl)triazene] binding to DNA duplexes and to a RNA duplex: evidence for both intercalative and minor groove binding properties.

Berenil is an antitrypanosomal agent that binds to nucleic acid duplexes. The generally accepted mode of berenil binding is via complexation into the minor groove of AT-rich domains of DNA double helices. We find that berenil can bind to RNA as well as DNA duplexes, while exhibiting properties characteristic of both intercalation as well as minor groove binding. More specifically, we use spectroscopic, calorimetric, and hydrodynamic techniques to characterize berenil binding to four DNA duplexes and to one RNA duplex. Our results reveal the following features: (i) Berenil binding to the poly[d(A-T)]2, poly(dA).poly(dT), poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes exhibits intercalative as well as minor groove binding characteristics. (ii) The apparent "site sizes" associated with berenil binding to these five duplexes range from 1 to 13 base pairs per bound berenil and depend, in part, on the host duplex. One of the site sizes common to all five duplexes is consistent with berenil binding to the minor groove. (iii) The apparent berenil binding affinity follows the hierarchy: poly(dA).poly(dT) > poly-[d(A-T)]2 approximately poly[d(I-C)]2 >> poly(rA).poly(rU) > poly[d(G-C)]2. (iv) Viscometric data reveal properties characteristic of a significant contribution from an intercalative mode of binding when berenil interacts with the poly[d(A-T)]2, poly[d(I-C)]2, poly[d(G-C)]2, and poly(rA).poly(rU) duplexes, while revealing an apparent nonintercalative mode when the drug binds to the poly(dA).poly(dT) duplex. (v) Berenil binding unwinds negative supercoils in the pBR322 plasmid, an observation consistent with an intercalative mode of binding to duplex DNA. (vi) Salt-dependent melting data suggest that both positively charged amidino groups of berenil participate in the complexation of the drug to the poly[d(I-C)]2, poly[d(A-T)]2, poly(dA).poly(dT), and poly(rA).poly(rU) duplexes, while also suggesting that the binding event is site-specific. In the aggregate, our results suggest that, in contrast to the conventional wisdom, berenil can exhibit intercalative as well as minor groove binding properties when it binds to both DNA and RNA duplexes, while also exhibiting a preference for DNA duplexes with unobstructed minor grooves. We comment on the potential correlation between drugs, such as berenil, that exhibit "mixed" binding motifs and those that express anticancer activity via inhibition of topoisomerase I activity.

Binding Sites↗

PubMed1995

Helix-stabilizing compounds CC-1065 and U-71,184 bind to RNA-DNA and DNA-DNA duplexes containing modified internucleotide linkages and stabilize duplexes against thermal melting.

CC-1065 and U-71,184 bind and hyperstabilize DNA duplexes, but little is known about their effects on nucleic acid duplexes of different structure. A 20 mer DNA sequence (5'-TTACTTCAGTTATGAGACCA) containing a drug binding sequence (5'-AGTTA) was selected as the target sequence, and this was duplexed with complementary antisense sequences containing phosphodiester (PO), phosphorothioate (PS), and methylphosphonate (MP) bonds. The duplexes containing PO or PS bound 2 CC-1065 molecules per duplex, presumably at both the target site and at a lower affinity site (5'-AGTAA) on the antisense strand. The duplex containing MP bound only 1 CC-1065, and all duplexes bound only 1 U-71,184. Both CC-1065 and U-71,184 bound to 20 mer duplexes comprised of oligo(dA)-oligo(dT) (2.5 and 2 drugs per duplex, respectively) and poly(rA)-oligo(dT) (1 drug per 20 base pairs). CC-1065 also bound to duplexes between the PO- or PS-based antisense structures and a complementary synthetic 20 mer RNA sequence, with about 1 drug per duplex in each case. CC-1065 increased the Tm for the 20 mer DNA duplexes 17 to 29 degrees C, and the corresponding values for U-71,184 ranged from 7 to 19 degrees C. CC-1065 raised the Tm of oligo(dA)-oligo(dT) and poly(rA)-oligo(dT) 29 degrees C. U71,184 increased the Tm for oligo(dA)-oligo(dT) 30 degrees C but did not significantly elevate the Tm for the corresponding RNA-DNA duplex. The results show that CC-1065 and U-71,184 are capable of binding and stabilizing a variety of nucleic acid duplexes. These agents or their analogs may become useful ligands for antisense oligonucleotide applications.

Antineoplastic Agents↗

PubMed2004

Value of ultrasonographic contrast in duplex scanning of leg arteries. Comparison with conventional duplex scanning and arteriography.

AIM: Duplex scanning has been used in the evaluation of the aorta and proximal arteries of the lower extremities, but has limitations in evaluating the arteries of the leg. The utilization of ultrasonographic contrast (USC) may be helpful in improving the quality of the image in these arteries. The objective of the present study was to verify whether the USC increases the diagnostic accuracy of patency of the leg arteries and if it diminishes the time needed to perform duplex scanning. METHODS: Twenty patients with critical ischemia (20 lower extremities) were examined by standard duplex scanning, duplex scanning with contrast and digital subtraction arteriography (DSA). The 3 arteries of the leg were divided into 3 segments, for a total of 9 segments per limb. Each segment was evaluated for patency in order to compare the 3 diagnostic methods. Comparison was made between standard duplex scanning and duplex scanning with contrast in terms of quality of the color-coded Doppler signal and of the spectral curve, and also of the time to perform the exams. RESULTS: Duplex scanning with contrast was similar to arteriography in relation to patency diagnosis (p>0.3) and even superior in some of the segments. Standard duplex scanning was inferior to arteriography and to duplex scanning with contrast (p<0.001). There were improvements of 70% in intensity of the color-coded Doppler signal and 76% in the spectral curve after the utilization of contrast. The time necessary to perform the examinations was 23.7 minutes for standard duplex scanning and 16.9 minutes for duplex scanning with contrast (p<0.001). CONCLUSIONS: The use of ultrasonographic contrast increased the accuracy of the diagnosis of patency of leg arteries and diminished the time necessary for the execution of duplex scanning.

Adult↗

PubMed2005

Unique properties of purine/pyrimidine asymmetric PNA.DNA duplexes: differential stabilization of PNA.DNA duplexes by purines in the PNA strand.

PNA.DNA duplexes are significantly stabilized by purine nucleobases in the PNA strand. To elucidate and understand the effect of switching the backbone in a nucleic acid duplex, we now report a thermodynamics study along with a solution conformations study of two purine/pyrimidine strand asymmetric duplexes and a strand symmetrical control by comparing the behavior of all four possible PNA/DNA combinations. In essence, we are comparing an identical basepair stack connected by either an aminoethyl glycine PNA or a deoxyribose DNA backbone. We show that the PNA.DNA duplexes containing purine-rich PNA strands are stabilized with regard to the thermal melting temperature and free energy as well as enthalpy (and concomitantly relatively less entropically disfavored). Based on our data, we find it unlikely that differences in counterion binding (identical ionic-strength dependence was observed), hydration (identical and insignificant water release was observed), or single-strand conformation can be responsible for the difference in duplex stability. The only consistent difference observed between the purine-rich PNA versus the pyrimidine-rich PNA in isosequential PNA.DNA duplexes is the significant increase in both binding enthalpy and entropy for the PNA.DNA duplexes containing pyrimidine-rich PNA in organic solvent, which would indicate that these duplexes are relatively enthalpically disfavored in water. Although our results so far do not allow us to identify the origin of the different stabilities of homopurine/homopyrimidine PNA.DNA duplexes, the evidence does point to a significant structural component, which involves enthalpic contributions both within the duplex structure and also from bound water molecules.

Base Composition↗

PubMed1992

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.

Base Sequence↗

PubMed1995

Base-pair exchange kinetics of the imino and amino protons of the 3'-phenazinium tethered DNA-RNA duplex, r(5'GAUUGAA3'):d(5'TCAATC3'-Pzn), and their comparison with those of B-DNA duplex.

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [5'r(G1A2U3U4G5A6A7)3']:5'p[d(T8C9A10A11T12C13)]3'-Pzn ] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C1G2T3A4C5G6)2 (II), d[p(5'T1G2T3T4T5G6G7C8)3']:d[p(5'C9C10A11A12A13C14A15)3'] (III), d(C5G6C7G8A9A10T11T12C13G14C15G16)2 (IV) and d(C1G2C3G4C5G6C7G8A9A10T11T12C13G14C15G16C17G18C19G20)2 (V). This comparative study shows that the life-times tau o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of approximately 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the tau o of the larger duplexes (IV) and (V): the tau o for the base pair of T11 and T12 residues in the 20-mer (duplex V) are 2.9 +/- 2.3 ms and 23.2 +/- 8.9 ms, respectively, while the corresponding tau o in the 12-mer (duplex IV) are 2.8 +/- 2.2 ms and 17.4 +/- 5.4 ms. It has also been shown that the total energy of activation (Ea) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the Ea of the short B-DNA duplex (Ea approximately 28-47 kcal/mol).

Base Composition↗

PubMed2001

The first example of a Hoogsteen base-paired DNA duplex in dynamic equilibrium with a Watson-Crick base-paired duplex--a structural (NMR), kinetic and thermodynamic study.

A single-point substitution of the O4' oxygen by a CH2 group at the sugar residue of A6 (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C1G2C3G4A5A6T7T8C9G10C11G12)2(-3), has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)<==>(1B): Keq = k1/k(-1) = 0.56+/-0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9 0.8 sec(-1); deltaHdegrees++ = 164+/-14 kJ/mol; -TdeltaS degrees++ (298K) = -92 kJ/mol giving a deltaG degrees++ 298 of 72 kJ/mol. Ea (k1) = 167 14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 0.6 sec-1, deltaH degrees++ = 153 13 kJ/mol; -TdeltaSdegrees++ (298K) = -82 kJ/mol giving a deltaGdegrees++(298) of 71 kJ/mol. Ea (k-1) = 155 13 kJ/mol]. Acomparison of deltaGdegrees++(298) of the forward (k1) and backward (k-1) conversions, (1A)<==>(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A6, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in alpha, sigma and gamma torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

Base Pairing↗

PubMed1995

DNA duplexes flanked by hybrid duplexes: the solution structure of chimeric junctions in [r(cgcg)d(TATACGCG)]2.

Hybrid duplexes and chimeric duplexes containing hybrid segments linked to pure DNA (or pure RNA) segments are involved in transcription and replication, as well as reverse transcription. A complete understanding of the mechanism of these processes requires detailed information on such duplexes and the junctions between duplexes of differing structure. Using two-dimensional NMR, restrained molecular dynamics and mechanics, and back-calculation refinement against the nuclear Overhauser effect spectra at various mixing times, we have determined the solution structure of the chimeric duplex [r(cgcg)d-(TATACGCG)]2 containing a pure DNA segment in the center of a hybrid duplex. The solution structure differs from the previously determined X-ray structure of the analogous duplex [r(gcg)d(TATACGC)]2, which was found to be A-form throughout [Wang, A.H.-J., et al. (1982) Nature 299, 601-604]. The basic features of the solution structure are (a) the RNA residues are all A-form with C3'-endo sugar conformations, (b) the central DNA segment is B-form, (c) the transition from A-form RNA sugar conformations to B-form DNA sugar conformations involves only the dT5 base step, and (d) although the sugar conformations of the DNA residues A6-G12 are closer to B-form, the basic helical properties of the peripheral RNA.DNA hybrid segments are closer to typical A-form than to B-form.

Base Sequence↗

PubMed1989

Two-dimensional 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex and a quinoxaline ((MeCys3, MeCys7)(TANDEM) drug duplex complex.

Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine "rules" previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only small perturbations in the 1H NMR and UV spectra are observed upon binding the drug to the decamer, although association of the drug stabilizes the duplex form relative to the other states. These results are consistent with a non-intercalative mode of association of the drug. Modeling and molecular mechanics energy minimization demonstrate that a novel structure in which the two quinoxaline rings of the drug binds in the minor groove of the duplex is possible.

Animals↗

PubMed2002

Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs.

A DNA fragment d(GCGAAAGCT), known to adopt a stable mini-hairpin structure in solution, has been crystallized in the space group I4(1)22 with the unit-cell dimensions a = b = 53.4 A and c = 54.0 A, and the crystal structure has been determined at 2.5 A resolution. The four nucleotide residues CGAA of the first half of the oligomer form a parallel duplex with another half through the homo base pairs, C2:C2+ (singly-protonated between the Watson- Crick sites), G3:G3 (between the minor groove sites), A4:A4 (between the major groove sites) and A5:A5 (between the Watson-Crick sites). The two strands remaining in the half of the parallel duplex are split away in different directions, and they pair in an anti-parallel B-form duplex with the second half extending from a neighboring parallel duplex, so that an infinite column is formed in a head-to-tail fashion along the c-axis. It seems that a hexa-ammine cobalt cation supports such a branched and bent conformation of the oligomer. One end of the parallel duplex is stacked on the corresponding end of the adjacent parallel duplex; between them, the guanine base of the first residue is stacked on the fourth ribose of another duplex.

Base Pairing↗

PubMed2005

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference.

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene-DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. (1)H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene-RNA/RNA and the pyrene-DNA/DNA duplexes.

Circular Dichroism↗

PubMed2004

Crystal structure of d(CGAAGC); association of two parallel-stranded duplexes through anti-parallel-stranded duplex formation.

We reported that d(CGAA) prefers to form a parallel duplex with homo base pair formations. To survey a possibility of longer parallel duplex, crystal structure of d(CGAAGC) has been determined. As expected, the CGAA part of the hexamer forms a parallel duplex with the corresponding part of another hexamer. The remaining G and C residues of one strand are stacked on the end of the parallel duplex, while those of the other strand are flip out and extended to the outside. The stacked residues form an anti-parallel duplex with the extended residues of the adjacent parallel duplex to form a dimer between the two parallel duplexes.

Base Sequence↗

PubMed2004

Measurement of nucleobase pKa values in model mononucleotides shows RNA-RNA duplexes to be more stable than DNA-DNA duplexes.

To understand why the RNA-RNA duplexes in general has a higher thermodynamic stability over the corresponding DNA-DNA duplexes, we have measured the pK(a) values of both nucleoside 3',5'-bis-ethyl phosphates [Etp(d/rN)pEt] and nucleoside 3'-ethyl phosphates [(d/rN)pEt] (N = A, G, C, or T/U), modeling as donors and acceptors of base pairs in duplexes. While the 3',5'-bis-phosphates, Etp(d/rN)pEt, mimic the internucleotidic monomeric units of DNA and RNA, in which the stacking contribution is completely absent, the 3'-ethyl phosphates, (d/rN)pEt, mimic the nucleotide at the 5'-end. The pK(a) values of the nucleobase in each of these model nucleoside phosphates have been determined with low pK(a) error (sigma = +/-0.01 to 0.02) by (1)H NMR (at 500 MHz) with 20-33 different pH measurements for each compound. This study has led us to show the following: (1) All monomeric DNA nucleobases are more basic than the corresponding RNA nucleobases in their respective Etp(d/rN)pEt and (d/rN)pEt. (2) The pK(a) values of the monomeric nucleotide blocks as well as Delta pK(a) values between the donor and acceptor can be used to understand the relative base-pairing strength in the oligomeric duplexes in the RNA and DNA series. (3) The Delta G*(pKa) of the donor and acceptor of the base pair in duplexes enables a qualitative dissection of the relative strength of the base-pairing and stacking in the RNA-RNA over the DNA-DNA duplexes. (4) It is also found that the relative contribution of base-pairing strength and nucleobase stacking in RNA-RNA over DNA-DNA is mutually compensating as the % A-T/U content increases or decreases. This interdependency of stacking and hydrogen bonding can be potentially important in the molecular design of the base-pair mimics to expand the alphabet of the genetic code.

DNA↗

PubMed2026

Duplex-Indel: a Snakemake pipeline for somatic Indel calling in Tn5 transposase-based duplex sequencing data.

SUMMARY: Duplex-Indel is a novel Snakemake workflow for detecting somatic small insertions and deletions (Indels) from Tn5 transposase-based duplex sequencing data. Duplex-Indel enhances the accuracy of mutation calling at the single-molecule level by requiring consensus support from both DNA strands for each somatic Indel, minimizing confounding from technical artifacts. Duplex-Indel extends somatic mutation calling in Tn5 transposase-based duplex sequencing data to include Indels. We have demonstrated the accuracy and robustness of Duplex-Indel using cancer cell lines. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are available under the MIT license on GitHub at https://github.com/ealee-lab/duplex-indel and archived on Zenodo at https://doi.org/10.5281/zenodo.19228799.

Transposases↗