Endcaps for stabilizing short DNA duplexes

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.     

Structure and Conformation of the Duplex Consensus Acceptor Exon: Intron Junction d [(CpTpApCpApGpGpT) (ApCpCpTpGpTpApG)] deduced from High-Field 1H-NMR of Non-Exchangeable and Imino Protons

Abstract

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20°C as evidenced by temperature variable ¹H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and ¹H-¹H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D₂O:H₂O mixtures. Assignment of the nonexchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5′ regions exist predominantly in the S form (2′endo—3′exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2′exo—3′endo). The contiguous bases A₅-G₆ (adjacent to the junction) and A₁₅-G₁₆ are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.     

Sequence Specific Molecular Recognition and Binding of a Monocationic Bis-Imidazole Lexitropsin to the Decadeoxyribonucleotide d-[(GATCCGTATG) (CATACGGATC)]: Structural and Dynamic Aspects of Intermolecular Exchange Studied by 1H-NMR

Abstract

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG) · (GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2′-endo-3′-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH₃(7). The base, anomeric H1′ and imino proton signals for the base sequence 5′-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5′-CCGT in the minor groove of the DNA NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the amino terminus oriented to C(4). The specific binding of 1 to the sequence 5′-CCGT-3′ deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci I fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA The [(T₇A₈T₉)· (A₁₂T₁₃A₁₄)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5′-CCGT sites, at a concentration of 12 mM, is estimated to be ～88s^?1 at 308°K with ΔG≠ of 63±5 K.J mol^?1.     

1H NMR Structural Analysis of the Interactions of Proflavine with Self-Complementary Deoxytetranucleosides of Different Base Sequence

Abstract

The molecular associations and structures of the complexes between the acridine dye, proflavine, and self-complementary deoxytetraribonucleoside triphosphates 5′-d(GpCpGpC), 5′-d(CpGpCpG), 5′-d(ApCpGpT), 5′-d(ApGpCpT) in aqueous solution have been investigated using one-dimensional and two-dimensional 500 MHz ¹H NMR spectroscopy.     

Synthesis of Dumbbell-Shaped Circular DMA-Molecules

Abstract

The syntheses of two dumbbell-shaped circular DMA-Molecules of chain lengths 140 and 150 nt are described. They are formed by enzymic ligation of chemically synthesized oligonucleotides containing self-complementary 5′ protruding ends.     

An NMR Study of the Polymorphous Behavior of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G) in Solution. The B,Z, and Hairpin Forms

Abstract

The polymorphism exhibited by the mismatched octamer d(m ⁵C-G-m⁵C-G-T-G-m⁵C-G), as a function of the temperature, DNA concentration and ionic strength, was investigated by means of NMR spectroscopy.

It is shown that this partly self-complementary DNA fragment, under conditions of low DNA concentration (0.4 mM) and low ionic strength, exclusively prefers to adopt a monomeric hairpin form, which consists of a stem of three Watson-Crick-type base pairs and a loop of only two residues. This in striking contrast with earlier intimations in literature, which postulated that in oligonucleotides loop formations containing only two residues are sterically impossible. Moreover, the hairpin form displays an unusual stability in comparison with previously reported hairpins. AT_m of 332 K and a ΔH° of—130 kj · mol^?1 were calculated for the hairpin to random coil transition.

At high DNA concentration (8 mM)and/or upon the addition of sodium chloride the hairpin form occurs in slow exchange with a B-DNA dimer structure (approximately 20% at 270 K, no added salt), which comprises two central GxT-mismatched base pairs with the bases as major tautomers.

At higher ionic strength (> 100 mM NaCI), or upon the addition of methanol, a third species appears, which is in slow exchange with both the B dimer and the hairpin form. This third species could be identified with a Z DNA form, comprising two GxT mismatches with the bases as major tautomers, with the guanine bases syn and the cytosine and thymine bases anti.     

Nucleosides,LVIII1 Synthesis of Base - Modified Oligonucleotides Containing 6- and 7-Aryl Lumazines

Abstract

6-Phenyl-, 7-phenyl-, 6-(4-biphenyl)- 7-(4-biphenyl)lumazine N-1-2-deoxy-β-D-ribofuranosides were synthesized, then converted into the corresponding 5′-O-dimethoxytrityl-3′-O-(β-cyanoethyl, N,N-diisopropyl)phosphoramidites and incorporated into different positions of self-complementary oligonucleotides. The influence of modifications on the melting temparature of the resulting duplexes was studied.

     

Acyclic 2′, 3′-Dideoxy-2′,3′-didehydronucleoside 5′-Triphosphates as Termination Substrates of Broad Set of DNA Polymerases

Abstract

O4′-Nor-2′,3′-dideoxy-2′,3′-didehydronucleoside 5′-triphosphates (acyclo-d₄NTP) have been shown to have the properties of effective termination substrates for DNA biosynthesis, catalyzed by several different DNA polymerases.     

Explicit Solvent Molecular Dynamics Simulation of Duplex Formed by the Modified Oligonucleotide with Alternating Phosphate/Phosphonate Internucleoside Linkages and its Natural Counterpart

Abstract

Impact of the internucleoside linkage modification by inserting a methylene group on the ability of the modified oligonucleotide to hybridize with a natural DNA strand was studied by fully solvated molecular dynamics (MD) simulations. Three undecamer complexes were analyzed: natural dT₁₁.dA₁₁ duplex as a reference and two its analogs with alternating modified and natural linkages in the deoxyadenosine chain. The isopolar, non-isosteric modified linkages were of 5′-O-PO₂-CH₂-O-3′ (5′PC3′) or 5′-O-CH₂-PO₂-O-3′ (5′CP3′) type. Simulations were performed by using the AMBER 5.0 software package with the force field completed by a set of parameters needed to model the modified segments. Both modifications were found to lead to double helical complexes, in which the thymidine strand as well as deoxyriboses and unmodified linkages in the adenosine strand adopted conformations typical for the B-type structure. For each of the two conformational richer modified linkages two stable conformations were found at 300 K: the -ggt and ggt for the 5′PC3′ and ggg, tgg for the 5′CP3′, respectively. Both modified chains adopted helical conformations with heightened values of the inclination parameter but without affecting the Watson-Crick hydrogen bonds.     

Fluorescence Properties and Base Pair Stability of Oligonucleotides Containing 8-Aza-7-deaza-2′-deoxyisoinosine or 2′-Deoxyisoinosine

Abstract

The fluorescence and the base pairing properties of 8-aza-7-deaza-2′-deoxyisoinosine (1) are described and compared with those of 2′-deoxyisoinosine (2). The corresponding phosphoramidites (11,12) are synthesized using the diphenyl-carbamoyl (DPC) residue for the 2-oxo group protection. The nucleosides 1 and 2 base pair with 2′-deoxy-5-methylisocytidine in DNA duplexes with antiparallel chain orientation and with 2′-deoxycytidine in a parallel DNA. These base pairs are less stable than the canonical dA-dT pair and that of 2′-deoxyinosine (4) with 2′-deoxycytidine. The fluorescence of the nucleosides 1 and 2 is quenched (～95%) in duplex DNA. The residual fluorescence is used to determine the T_m-values, which are found to be the same as determined UV-spectrophotometrically.     

The Parallel and Antiparallel Triplex Formation and Stability of Self Complementary Oligonucleotides Containing 2′-Fluoro-arabinosyl Thymine and 5-Methyl-2′-Deoxycytidine

Abstract

We examined the effects of 1–(2-deoxy -2-fluoro-β-D-arabinofuranosyl)-thymine (or FMAU, a potent antiviral nucleoside) on the stability of duplex and triplexes. When compared the stability of the self-complementary 5′-A₅T₅ duplex with 5′-A₅X₅ (X = FMAU), duplex containing FMAU has much higher melting temperature (T_m). 5′-A₆T₅T₃X₃T₅F₃X₃ and T₃X₃T₅A₆T₅F₃X₃ form the parallel and antiparallel triplexes T₃X₃: A₆:X₃X₃, respectively. The former exhibited the typical T:A:T triplex behavior with only one melting temperature at 70 °C and 45 °c in 1.0 M and 0.2 M NaCl solution, respectively, whereas the latter has two T_m values at 56 °C and 28 °C in 1.0 M solution. FMAU clearly stabilize the triplex structure as A₆T₂₂ which forms the parallel triplex T₆:A₆:T₆ has also only one T_m at 54 °C and 37 °C in high and iow salt concentration solutions, respectively. A 31mer 5′-TCCTCCTTTTTTAGGAGGATTTTTTGGTGGT and 5′-TCCTCCTTTTTTAGGAGGATTTTTTX'X'TX'X'T (X' = 2′-deoxy-5-methylcytidine) were prepared to study their triplex forming potential. The former was found to have a week interaction of the Watson-Crick duplex with the mismatched third-strand at all pH. The latter formed a stable triplex at lower pH consistent with required protonation on the 5-methylcytosine base. For these studies we developed a simple PC desktop spreadsheet program to calculate the first derivative profile of the melting curve data.

This paper is dedicated to Prof. Jacques H. van Boom on the occasion of his 60th birthday.     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

Conformational and Model-Building Studies of the Hairpin Form of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G)

Abstract

The hairpin form of the mismatched octamer d(m⁵C-G-m⁵C-G-T-G-m⁵C-G) was studied by means of NMR spectroscopy. In a companion study it is shown that the hairpin form of this DNA fragment consists of a structure with a stem of three Watson-Crick-type base pairs and a loop consisting of only two nucleotides. The non-exchangeable proton resonances were assigned by means of two-dimensional correlation spectroscopy and two-dimensional nuclear Overhauser effect spectroscopy. Proton-proton coupling constants were used for the conformational analysis of the deoxyribose ring and for some of the backbone torsion angles. From the two-dimensional NMR spectra and the coupling-constant analysis it is concluded that: (i) the stem of the hairpin exhibits B-DNA characteristics; (ii) the sugar rings are not conformationally pure, but display a certain amount of conformational flexibility; (iii) the stacking interaction in the stem of the hairpin is elongated from the 3′-side in a more or less regular fashion with the two loop nucleotides; (iv) at the 5′-side of the stem a stacking discontinuity occurs between the stem and the loop; (v) at the 5′-side of the stem the loop is closed by means of a sharp backbone turn which involves unusual γ^t and β⁺ torsion angles in residue dG(6).

The NMR results led to the construction of a hairpin-loop model which was energy-minimized by means of a molecular-mechanics program. The results clearly show that a DNA hairpin-loop structure in which the loop consists of only two nucleotides bridging the minor groove in a straightforward fashion, (i) causes no undue steric strain, and (ii) involves well-known conformational principles throughout the course of the backbone.

The hairpin form of the title compound is compared with the hairpin form of d(A-T-C-C-T- A-T₄-T-A-G-G-A-T), in which the central -T₄- part forms a loop of four nucleotides. Both models display similarities as far as stacking interactions are concerned.     

NMR Studies of Tris-Intercalation: Solution Structure and Interaction of d(CTTCGCGCGAAG) with an Acridine Trimer

Abstract

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of ¹H and ³¹P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H₅', H_5”), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O_1′ endo range, those of the internal guanosines being closer to C_2′endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the ³T⁴C⁵G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3′-5′)G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

Synthesis and Biological Activities of Sugar-Modified 2-(p-n-Butylanilino)-2′-deoxyadenosine Analogues

Abstract

Several sugar-modified 2-(p-n-butylanilino)-2′-deoxyadenosine analogues, including arabino and 2′(R)-azido-2′-deoxy analogues and their 5′-triphosphates were synthesized. These nucleosides thus obtained exhibited moderate cytotoxicity against P-388 leukemic cells in culture (IC₅₀ = 13–24 μ). In contrast to above results, the 5′-triphosphates have been shown to exert strong and selective inhibitory effects on mammalian DNA polymerase α (Ki= 0.02–0.04 μ).     

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

Abstract

A single-point substitution of the O4′ oxygen by a CH₂ group at the sugar residue of A ⁶ (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 A ⁶:T⁷ Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): K_eq = 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; δH°? = 164±14 kJ/mol;-TδS°? (298K) = ?92 kJ/mol giving a δG₂₉₈°? 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, δH°? = 153±13 kJ/mol;-TδS°? (298K) = ?82 kJ/mol giving a δG₂₉₈°? of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG₂₉₈°? 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 A ⁶: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 A ⁶:T⁷ basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A ⁶, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A ⁶. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.     

Synthesis,Biophysical, and Biochemical Properties of PNA-DNA Chimeras

Abstract

Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T₂₀-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA₂₀ and their hydrolyses by 3′- and 5′-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.     

Enhanced uranyl photocleavage across the minor groove of all (A/T)4 sequences indicates a similar narrow minor groove conformation

The uranyl(VI)-mediated photocleavage of a Drew–Dickerson sequence oligonucleotide (5′-dGATCACGCGAATTCGCGT) either as the (self-complementary) duplex or cloned into the BamH1 site of pUC19 has been studied. At pH 6.5 in acetate buffer relatively enhanced photocleavage is observed at the 3′-end of the AATT sequence corresponding to maximum cleavage across the minor groove in the A/T tract. Thus maximum cleavage correlates with minimum minor groove width in the crystal structure and also with the largest electronegative potential according to computations. Using plasmid constructs with cloned inserts of the type [CGCG(A/T₄)]_n, we also analysed all possible sequence combinations of the (A/T)₄ tract and in all cases we observed maximum uranyl-mediated photocleavage across the minor groove in the (A/T)₄ tract without any significant differences between the various sequences. From these results we infer that DNA double helices of all (A/T)₄ sequences share the same narrow minor groove helix conformation.     

Sensitivity of Bacteriophage RB69 DNA Polymerase to n2-(p-n-Butylphenyl)-2′-Deoxyguanosinenucleotides

Abstract

The response of bacteriophage RB69 DNA polymerase to N²-(p-n-butylphenyl)-2′-deoxyguanosine 5′-triphosphate (BuPdGTP), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related DNA polymerase from bacteriophage T4. Both enzymes showed similar responses to inhibitors in terms of K_i values and the ability to utilize BuPdGTP as a substrate. These results provide the relevance of using the recent crystal structure of RB69 DNA polymerase for analysis of BuPdGTP/B family DNA polymerase interactions.