Spectroscopic studies on the formation and thermal stability of DNA triplexes with a benzoannulated delta-carboline-oligonucleotide conjugate

A benzoannulated delta-carboline with a phenyl substituent has been covalently tethered to the 3'-end of a triplex-forming oligonucleotide and its ability to bind and stabilize DNA triple helices has been examined by various spectroscopic methods. UV thermal melting experiments were conducted with different hairpin duplexes and with a complementary single-stranded oligonucleotide as targets for the conjugate. The delta-carboline ligand preferentially binds triplexes over duplexes and leads to a temperature increase of the triplex-to-duplex transition by up to 23 degrees C. The results obtained from UV, CD and fluorescence measurements suggest that the delta-carboline ligand exhibits specific interactions with a triplex and favors binding by intercalation at the triplex-duplex junction.     

Design of phosphoramidite monomer for optimal incorporation of functional intercalator to main chain of oligonucleotide

Chirally pure phosphoramidite monomers bearing 9-amino-6-chloro-2-methoxyacridine were synthesized from D- or L-threoninol and omega-aminocarboxylic acid, and incorporated into oligonucleotides. These acridine-DNA conjugates formed stable duplexes with complementary RNA because of intercalation of the acridine to DNA/RNA heteroduplexes. The stability of duplexes was not very dependent on either the chirality of the central carbon bearing the acridine or the length of the side chain. However, the ability for site-selective activation of the phosphodiester linkage in front of the acridine, which induced Lu(III)-promoted RNA scission, was strongly dependent on these two factors. The largest activation was achieved when the monomer unit was prepared from L-threoninol and 4-aminobutyric acid and the acridine was bound to the amino group. By attaching the more acidic 9-amino-2-methoxy-6-nitroacridine to this optimized scaffold, a quite effective acridine-DNA conjugate for site-selective RNA scission was obtained.     

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (T_m) of the peptide–RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as l-2-amino-4-guanidinobutyric acid (Agb) or l-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the l-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide–RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.     

Synthesis and biological evaluation of new non-imidazole H3-receptor antagonists of the 2-aminobenzimidazole series

A novel series of non-imidazole H(3)-receptor antagonists was developed, by chemical modification of a potent lead H(3)-antagonist composed by an imidazole ring connected through an alkyl spacer to a 2-aminobenzimidazole moiety (e.g., 2-[[3-[4(5)-imidazolyl]propyl]amino]benzimidazole), previously reported by our research group. We investigated whether the removal of the imidazole ring could allow retaining high affinity for the H(3)-receptor, thanks to the interactions undertaken by the 2-aminobenzimidazole moiety at the binding site. The imidazole ring of the lead was replaced by a basic piperidine or by a lipophilic p-chlorophenoxy substituent, modulating the spacer length from three to eight methylene groups; moreover, the substituents were moved to the 5(6) position of the benzimidazole nucleus. Within both the 2-alkylaminobenzimidazole series and the 5(6)-alkoxy-2-aminobenzimidazole one, the greatest H(3)-receptor affinity was obtained for the piperidine-substituted compounds, while the presence of the p-chlorophenoxy group resulted in a drop in affinity. The optimal chain length was different in the two series. Even if the new compounds did not reach the high receptor affinity shown by the imidazole-containing lead compound, it was possible to get good H(3)-antagonist potencies with 2-aminobenzimidazoles having a tertiary amino group at appropriate distance.     

Long range molecular wire behaviour in a metal complex of DNA

M-DNA is a complex of metal ions such as Zn(2+) with duplex DNA. Previous results showed that the fluorescence of a donor fluorophore was quenched when an acceptor fluorophore was placed at the opposite end of a short M-DNA duplex. In order to investigate further the molecular wire behaviour of M-DNA, 30-mer duplexes were constructed with fluorescein as donor and rhodamine, pyrene and the cyanine dyes, Cy5 and Cy5.5 as acceptors. Good quenching was observed in all cases even though the efficiency of resonance energy transfer was calculated to be < 5%. The distance dependence of quenching was investigated by preparing doubly-labelled duplexes ranging in length from 20 to 1,000 base pairs. Upon formation of M-DNA significant quenching of the fluorescence of the donor fluorophore was observed in duplexes up to 500 base pairs in length. The amount of quenching decreased with increasing length of the duplexes with a shallow distance dependence. The results are consistent with an electron transfer mechanism in which the electron hops between metal centers. This process can occur efficiently over long distances.     

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.     

Influence of alkyl group on amide nitrogen atom on fluorescence quenching of tyrosine amide and N-acetyltyrosine amide

The steady-state and time-resolved fluorescence spectroscopy was applied to determine the influence of an alkyl substituent(s) (methyl or ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or t-butyl) on amide nitrogen atom on photophysical properties of tyrosine and N-acetyltyrosine amides in water. Generally, the amide group strongly quenches the fluorescence of tyrosine, however, the size and number of substituents on amide nitrogen atom modify the quenching process only in small degree. The fluorescence intensity decays of all amides studied are bi-exponential. The contribution of both components (alphai) to the fluorescence decay undergoes irregular change. An introduction of alkyl substituent on amide nitrogen atom causes an increase of the fluorescence lifetime of tyrosine derivative compared to the unsubstituted amide for both N-acetyltyrosine and tyrosine with the protonated amino group. Calculated, basing on the fluorescence quantum yield (QY) and average lifetime, the radiative rate constants (kf) are similar, which indicates that the substituent(s) does not have substantial influence on radiative process of the deactivation of the excited state of the phenol chromophore for all compounds studied regardless the amino group status as well as the number and type of substituent (linear or branched). The comparison of the ground-state rotamer populations of tyrosine amides and N-acetyltyrosine amides with different alkyl substituent on amide nitrogen atom obtained from 1H NMR with the value of pre-exponential factors indicates that not the rotamer populations, but specific hydration of a whole molecule of the amino acid including chromophore and amino acid moiety, seems to be the main reason of the heterogenous fluorescence intensity decay of tyrosine derivatives.     

Nitro and amino substitution within the A-ring of 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: influence on topoisomerase I-targeting activity and cytotoxicity

Recently, 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one, 1, was identified as a TOP1-targeting agent with pronounced antitumor activity. In the present study, the effect on activity of substituting a single nitro or amino group in the A-ring in lieu of the methylenedioxy moiety of 1 was evaluated. The presence of either a nitro or amino substituent at the 4-position had a pronounced adverse affect on both TOP1-targeting activity and cytotoxicity. To a lesser extent, derivatives with a nitro or amino substituent at the 1-position were also less active than 1. Replacement of the methylenedioxy moiety of 1 with either a nitro or amino substituent at either the 2- and 3-position did result in analogues with potent TOP1-targeting activity and cytotoxicity.     

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites. Probing of modified DNA duplexes as activators of the enzyme.

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11-14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiencies or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.     

Tris-benzimidazole derivatives: design, synthesis and DNA sequence recognition

Two tris-benzimidazole derivatives have been designed and synthesized based on the known structures of the bis-benzimidazole stain Hoechst 33258 complexed to short oligonucleotide duplexes derived from single crystal X-ray studies and from NMR. In both derivatives the phenol group has been replaced by a methoxy-phenyl substituent. Whereas one tris-benzimidazole carries a N-methyl-piperazine at the 6-position, the other one has this group replaced by a 2-amino-pyrrolidine ring. This latter substituent results in stronger DNA binding. The optimized synthesis of the drugs is described. The two tris-benzimidazoles exhibit high AT-base pair (bp) selectivity evident in footprinting experiments which show that five to six base pairs are protected by the tris-benzimidazoles as compared to four to five protected by the bis-benzimidazoles. The tris-benzimidazoles bind well to sequences like 5'-TAAAC, 5'-TTTAC and 5'-TTTAT, but it is also evident that they can bind weakly to sequences such as 5'-TATGTT-3' where the continuity of an AT stretch is interrupted by a single G*C base pair.     

Binding of 9-aminoacridine to bulged-base DNA oligomers from a frame-shift hot spot

Complexes of 9-aminoacridine and two derivatives with oligomers based on the sequence of a hot spot for frame-shift mutations, 5'dGATGGGGCAG, are investigated by proton NMR and equilibrium dialysis. Competition dialysis experiments show that the drug binds bulge-containing oligomers more strongly than regular duplexes of similar sequence and length, with one apparent strong site. A duplex containing an extra cytidine in a run of C's has the highest affinity for 9-aminoacridine among the sequences tested. An oligomer containing five consecutive G.C pairs shows cooperative drug binding, indicating that G tracts of this length may have an altered helical structure. Complexes of a regular 8-mer and a 9-mer containing a bulged guanosine are examined in detail by two-dimensional NMR techniques. 9-Aminoacridine preferentially binds at TpG sites in the 8-mer but binds primarily at the bulged guanosine in the G-bulge 9-mer. Drug-DNA NOE's in the 8-mer complex are compared with the crystal structure of 9-aminoacridine and 5-iodo-CpG [Sakore et al. (1979) J. Mol. Biol. 135, 763-785]. The NMR data suggest that the drug intercalates across the base pairs of both strands with the amino group projecting into the minor groove.     

N(4)C-alkyl-N(4)C cross-linked DNA: bending deformations in duplexes that contain a -CNG- interstrand cross-link

Short DNA duplexes containing a 1,3-N(4)C-alkyl-N(4)C interstrand cross-link that joins the two C residues of a -CNG- sequence were prepared using either a phosphoramidite or convertible nucleoside approach. The alkyl cross-link consists of 2, 4, or 7 methylene groups. The duplexes, which contain a seven-base-pair core and A(3)/T(3) complementary 3'-overhanging ends, were characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Ultraviolet thermal denaturation studies showed that the duplexes denature in a cooperative manner and that the length of the cross-link affects the thermal stability. Thus, the transition temperature of the ethyl cross-linked duplex, 42 degrees C, is 16 degrees C higher than the melting temperature of the corresponding non-cross-linked control, whereas the transition temperatures of the butyl and heptyl cross-linked duplexes, 73 and 72 degrees C, respectively, are 46-47 degrees C higher. The reduced molecularity of denaturation of the cross-linked duplexes versus melting of the non-cross-linked duplex most likely accounts for these differences. Examination of molecular models suggests that the ethyl cross-link is too short to span the distance between the two C residues at the site of the cross-link in B-form DNA without causing distortion of the helix, whereas less and no distortion would be expected for the butyl and heptyl cross-links, respectively. The circular dichroism spectra, which show greatest deviation in the ethyl cross-linked duplex from B-form DNA, are consistent with this expectation. Anomalous mobilities on native polyacrylamide gels of multimers produced by self-ligation of each of the cross-linked duplexes suggest that the ethyl and butyl cross-linked duplexes undergo bending deformations, whereas multimers derived from the heptyl cross-linked duplex migrated normally. The bending angle was estimated to be 20 degrees, 13 degrees, and 0 degrees for the ethyl, butyl, and heptyl cross-linked duplexes, respectively. Thus, it appears that the degree of bending in these N(4)C-alkyl-N(4)C cross-linked duplexes is controlled by the length of the cross-link.     

Use of crosslinking for revealing the DNA phosphate groups forming specific contacts with the E. coli Fpg protein

Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time. Reactive internucleotide groups were introduced in dsDNA immediately 5' or 3' to the oxidative lesion and one or two nucleotides 5' or 3' away from it. We showed that the Fpg protein specifically binds to the modified DNA duplexes. The binding efficiency varied with the position of the reactive group and was higher for the duplexes containing substituted pyrophosphate groups at the ends of pentanucleotide with the oxoG in the center. The nicking efficiency of the DNA duplexes containing the reactive groups one or two nucleotides 5' away from the lesion was higher as compared to non-modified DNA duplex bearing only the oxidative damage. We found two novel non-hydrolizable substrate analogs for the Fpg protein containing pyrophosphate and substituted pyrophosphate groups 3' adjacent to the oxoG. Using crosslinking, we revealed the phosphate groups, 3' and 5' adjacent to the lesion, which have specific contacts with nucleophilic amino acids from the E. coli Fpg protein active center. The crosslinking efficiency achieved 30%. The approaches developed can be employed in the studies of pro- and eucaryotic homologs of the E. coli Fpg protein as well as other repair enzymes.     

O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.C pairing in dodecanucleotide duplexes

The pairing of O6etG with C located four base pairs in from either end of the self-complementary d(C1-G2-C3-O6etG4-A5-G6-C7-T8-C9-G10-C11-G12) duplex (designated O6etG.C 12-mer) has been investigated from an analysis of proton and phosphorus two-dimensional NMR experiments. The structural consequences of increasing the alkyl group size were elucidated from a comparative study of the pairing of O6meG4 with C9 in a related sequence (designated O6meG.C 12-mer). The NMR parameters for both O6alkG-containing dodecanucleotides are also compared with those of the control sequence containing G4.C9 base pairs (designated G.C 12-mer). The NOE cross-peaks detected in the two-dimensional NOESY spectra of the O6alkG.C 12-mer duplexes in H2O solution establish that the O6etG4/O6meG4 and C9 bases at the lesion site stack into the helix between the flanking C3.G10 and A5.T8 Watson-Crick base pairs. The amino protons of C9 at the O6alkG4-C9 lesion site resonate as an average resonance at 7.78 and 7.63 ppm in the O6etG.C 12-mer and O6meG.C 12-mer duplexes, respectively. The observed NOEs between the amino protons of C9 and the CH3 protons of O6alkG4 establish a syn orientation of the O6-alkyl group with respect to the N1 of alkylated guanine. A wobble alignment of the O6alkG4.C9 base pair stablized by two hydrogen bonds, one between the amino group of C9 and N1 of O6alkG and the other between the amino group of O6alkG and N3 of C9, is tentatively proposed on the basis of the NOEs between the amino protons of C9 at the lesion site and the imino protons of flanking Watson-Crick base pairs. The proton and phosphorus chemical shift differences between the O6etG.C 12-mer and O6meG.C 12-mer duplexes are small compared to the differences between these O6alkG-containing duplexes and the control G.C 12-mer duplex.     

The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity

In order to establish more firmly the immunoregulatory effect of platelet factor 4 (PF4) and develop a means to provide material for possible clinical use, the nucleotide sequence for PF4 was synthesized utilizing a ligation strategy of six duplexes ranging from 27 to 43 base pairs in length. The individual oligodeoxynucleotides were synthesized on an automated system. The resultant gene segment (226 base pairs), which incorporated convenient HindIII and BamHI overhangs at the 5' and 3' ends, respectively, was cloned into the pIN-III-ompA-2-expression vector in Escherichia coli, affording a fusion protein of Mr = 8900 with 7 additional amino acids at the amino terminus and 4 at the carboxyl terminus and with aspartic acid rather than asparagine in position 47. The recombinant PF4 (rPF4) was purified by heparin-agarose affinity chromatography and reverse-phase high performance liquid chromatography. It reacted with a monoclonal mouse anti-human PF4 antibody on a Western blot and in an enzyme-linked immunosorbent assay. The rPF4 protein exhibited an immunoregulatory effect like that of human PF4 in its ability to reverse concanavalin A-induced immunosuppression in BALB/c mice.     

The reactivity of lactyl-oxythiamin implies the role of the amino-pyrimidine in thiamin catalyzed decarboxylation

It has previously been established that the deprotonated amino substituent of the pyrimidine of thiamin diphosphate (ThDP) acts as an internal base to accept the C2H of the thiazolium in ThDP-dependent enzymes. The amino group has also been implicated in assisting the departure of the aldehydic product formed after loss of CO₂ from ketoacid substrates. However, the potential role for the pyrimidine amino group in the key decarboxylation step has not been assessed. Oxythiamin contains a hydroxyl group in place of the pyrimidine amino group in thiamin, providing a basis for comparison of reactivity. Lactyl-oxythiamin (LOTh), the conjugate of pyruvic acid and oxythiamin was prepared by condensation of ethyl pyruvate and hydroxyl-protected oxythiamin followed by deprotection and acidic hydrolysis of the ethyl ester. The rate constants observed for the decarboxylation of LOTh in neutral and acidic solutions are about four times smaller than those for the corresponding compound that contains the amino group, lactylthiamin. The difference in reactivity is consistent with the amino group’s participation in facilitating the decarboxylation step by allowing a competitive addition pathway that produces bicarbonate and has implications for the corresponding enzymic reaction.     

The effect of a single boranophosphate substitution with defined configuration on the thermal stability and conformation of a DNA duplex

Substitution of one non-bridging oxygen in a natural phosphodiester internucleotide linkage with a borano (-BH3) group results in a chiral phosphorus center in boranophosphate. UV thermal melting profiles were recorded for DNA duplexes formed between a DNA 9-mer with either an Sp or a Rp boranophosphate linkage in the middle and the complementary DNA 9-mer, as well as for their unmodified parent duplex. The thermal stability of the DNA duplexes was in the order of normal > Sp borano > Rp borano. CD spectra of all three duplexes exhibited typical B-DNA profile, which closely resembled each other.     

Differential nucleotide excision repair susceptibility of bulky DNA adducts in different sequence contexts: hierarchies of recognition signals

The structural origin underlying differential nucleotide excision repair (NER) susceptibilities of bulky DNA lesions remains a challenging problem. We investigated the 10S (+)-trans-anti-[BP]-N(2)-2'-deoxyguanosine (G*) adduct in double-stranded DNA. This adduct arises from the reaction, in vitro and in vivo, of a major genotoxic metabolite of benzo[a]pyrene (BP), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, with the exocyclic amino group of guanine. Removal of this lesion by the NER apparatus in cell-free extracts has been found to depend on the base sequence context in which the lesion is embedded, providing an excellent opportunity for elucidating the properties of the damaged DNA duplexes that favor NER. While the BP ring system is in the B-DNA minor groove, 5' directed along the modified strand, there are orientational distinctions that are sequence dependent and are governed by flanking amino groups [Nucleic Acids Res.35 (2007), 1555-1568]. To elucidate sequence-governed NER susceptibility, we conducted molecular dynamics simulations for the 5'-...CG*GC..., 5'-...CGG*C..., and 5'-...TCG*CT... adduct-containing duplexes. We also investigated the 5'-...CG*IC... and 5'-...CIG*C... sequences, which contain "I" (2'-deoxyinosine), with hydrogen replacing the amino group in 2'-deoxyguanosine, to further characterize the structural and dynamic roles of the flanking amino groups in the damaged duplexes. Our results pinpoint explicit roles for the amino groups in tandem GG sequences on the efficiency of NER and suggest a hierarchy of destabilizing structural features that differentially facilitate NER of the BP lesion in the sequence contexts investigated. Furthermore, combinations of several locally destabilizing features in the hierarchy, consistent with a multipartite model, may provide a relatively strong recognition signal.