New mono- and bis-intercalating compounds between DNA base pairs

With the aim to obtain new bis-intercalating agents in DNA a series of compounds was prepared linking two identical tricyclic moieties (two 4,5'-8-trimethylpsoralen (TMP)) or different (one TMP and one pyrido[3,2-b]quinoline) through a hydrocarburic aminated flexible chai. Bis-psoralen-amines as well as psoralen-pyrydoquinolin-amines were obtained. For comparison the corresponding mono-psoralen-amines were also prepared condensing a TMP moiety with various hydrocarburic aminated chains. Melting profiles of the complexes between bis-psoralen-amines or psoralen-pyridoquinolin-amines and DNA evidence two different thermal transitions, which can be correlated with bis-intercalation which takes place, at least in part, inside duplex DNA. On the other hand mono-psoralen-amines evidenced only one thermal transition, in line with mono-intercalation. Bis-intercalating agents complexed with DNA, under UVA irradiation photoconjugate covalently to the macromolecule, even if to a lower extent in comparison with mono-intercalating agents. Moreover these bis-intercalating agents in the photoreaction with DNA form inter-strand cross-links; also in this case bis-intercalating agents are less active than mono-intercalating agents.     

The chemical stability and structure of borides

Summary The data point to the close relationship between the chemical stability of borides and their structure.The structure of borides determines the character and strength of the boron-boron and metal-boron bonds and the chemical stability of borides depends on them.In the hydrolytic decomposition of borides of these metals the main part is played by the strength of the boron-boron bond. The previously stated regularity with regard to the increase in chemical stability of borides with increase in the percentage of boron in the structures is therefore completely confirmed. The least stability is exhibited by borides with single boron atoms in the lattice and the greatest stability is shown by borides with octahedral complexes of boron in skeleton structures.We can evidently conclude that the regularity under consideration is also applicable in a general form to high-temperature oxidation, nltriding, and carbidization. However, in these cases there are a number of other factors affecting the chemical stability of the borides. These problems require a special study.     

Stability of RNA hairpins closed by wobble base pairs

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXANmAYCC, where XY is the wobble base pair, GU or UG, and the underlined loop sequences are three to eight nucleotides. A nearest-neighbor analysis indicates the free energy of loop formation is dependent upon loop size and closing base pair. Hairpin loops closed by UG base pairs are on average 1.3 kcal/mol less stable than hairpins closed by GU base pairs. The hairpin loops closed by UG have approximately the same stability as hairpin loops closed by AU/UA base pairs, while the loops closed by GU are approximately 0.7 kcal/mol more stable than hairpins loops closed by GC/CG base pairs. These results, combined with the model previously developed [Serra et al. (1997) Biochemistry 36, 4844] to predict the stability for hairpin loops closed by Watson-Crick base pairs, allow for the following model to predict the stability of hairpin loops: delta G degree 37L(n) = delta G degree 37iL(n) + delta G degree 37mm + 0.6 (if closed by AU, UA, or UB) - 0.7 (if closed by GU) - 0.7 (if first mismatch is GA or UU except for loops closed by GU). Here, delta G degree 37iL(n) is the free energy increment for initiating a loop of n nucleotides with a CG or GC pair, and delta G degree 37mm is the free energy for the interaction of the first mismatch with the closing base pair. For hairpin loops of n = 4-9, delta G037iL(n) is 4.9, 5.0, 5.0, 5.0, 4.9, and 5.5 kcal/mol, respectively. For hairpin loops of n = 3, delta G degree 37L(3) = +4.8 + 0.6 (if closed by AU, UA, or UG) kcal/mol. Thermodynamic parameters for hairpin formation in 1 M NaCl for 13 naturally occurring RNA hairpin sequences closed by wobble base pairs are reported. The model provides good agreement for both TM and delta G degree 37 for most hairpins studied. Thermodynamic values for five terminal mismatches adjacent to wobble base pairs are also reported.     

2'-Deoxyisoguanosine adopts more than one tautomer to form base pairs with thymidine observed by high-resolution crystal structure analysis

The questions of whether different tautomeric forms of nucleic acid bases exist to any significant extent in DNA, or what their possible roles in mutation may be, are under intense scrutiny. 2'-Deoxyisoguanosine (iG) has been suggested to have a propensity to adopt the enol form. Isoguanine (also called 2-hydroxyadenine) can be found in oxidatively damaged DNA generated from treating DNA with a Fenton-type reactive oxygen-generating system and is known to cause mutation. We have analyzed the three-dimensional structure of the DNA dodecamer d(CGC[iG]AATTTGCG) (denoted iG-DODE) by X-ray crystallography and NMR. The crystal structure of the iG-DODE complexed with the minor groove binder Hoechst 33342, refined to 1.4 A resolution, showed that the two independent iG.T base pairs in the dodecamer duplex adopt different (one in Watson-Crick and the other in wobble) conformations. The high-resolution nature of the structure also affords unprecedented clear information about the conformation and interactions of the Hoechst drug. The Hoechst 33342 binds in the narrow minor groove at the iGAATT site, with the N-methylpiperazine ring near the iG4.T21 base pair. Three hydrogen bonds are found between the NH of the Hoechst ligand and T-O2 DNA atoms. In solution, the two iG.T base pairs in iG-DODE predominantly are in the wobble form at 2 degreesC. At higher temperatures, another duplex form (likely involving the enol form of iG) is in slow exchange with the keto form and becomes significantly populated, reaching approximately 40% at 40 degreesC. Our data support the conclusion that iG pairs with T in a Watson-Crick configuration to a significant extent at physiological temperature (37 degreesC), which may explain the facile incorporation rate of T across from an iG during in vitro DNA replication.     

Highly selective binding of metal ions to thymine-thymine and cytosine-cytosine base pairs in DNA duplexes

Very specific binding of of Hg(II) and Ag(I) cations unexpectedly and significantly stabilizes the naturally occurring miss-base pairs, thymine-thymine and cytosine-cytosine, in DNA duplexes.     

Thermodynamics of nonsymmetric tandem mismatches adjacent to G.C base pairs in RNA

The thermodynamic stabilities and structures of a series of RNA duplexes containing nonsymmetric tandem mismatches in the context of , where are tandem mismatches, were studied by UV melting and imino proton NMR. The contribution of one mismatch to the free energy increment for tandem mismatch formation depends on the identity of the other mismatch. Imino proton NMR indicates that this is partly because the structure of a mismatch is dependent on the adjacent mismatch. The results suggest that differences in size, shape, and hydrogen bonding of the adjacent mismatches play important roles in determining loop stability. A model for predicting stabilities of all possible tandem mismatches is proposed based on these and previous results.     

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.     

Repair and mutagenic potency of 8-oxoG:A and 8-oxoG:C base pairs in mammalian cells

Replication of the oxidative lesion 8-oxo-7,8-dihydroguanine (GO) leads to the formation of both 8-oxo-7,8-dihydroguanine:adenine (GO:A) and 8-oxo-7,8-di-hydroguanine:cytosine (GO:C) pairs. The repair and mutagenic potency of these two kinds of base pairs were studied in simian COS7 and human MRC5V1 cells using the shuttle vector technology. Shuttle vectors carrying a unique GO residue opposite either a C or an A were constructed, then transfected into recipient mammalian cells. DNA repair resulting in G:C pairs and mutation frequency, were determined using resistance to digestion by the Ngo MI restriction enzyme for screening and DNA sequencing of suspect mutants. Results showed that the GO:C mismatch was well repaired since almost no mutations were detected in the plasmid progeny obtained 72 h after cell transfection. The GO:A pair was poorly repaired since only 32-34% of the plasmid progeny contained G:C whereas two thirds contained A:T at the original site. Repair kinetics measured with a non-replicating vector deleted by 13 bp at the SV40 replication origin, showed that GO:A was slowly repaired. Only 30% of the mispairs were corrected in 12 h. During this time 100% of the plasmids containing GO:A pairs were replicated as seen by the replication kinetics in a vector with an intact SV40 replication origin. These results show that, under our experimental conditions, replication is occurring before completion of DNA repair which explains the high mutagenic potency of the GO:A mispair.     

The 5' stem-loop from human U4 snRNA adopts a novel conformation stabilized by G-C and G-U base pairs

1H NMR and molecular modeling studies of the 5' stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5' side and a single bulged A on the 3' side. Both stems also had bulged A residues. 1H NMR experiments performed on solutions of the 35 mer stem-loop, the stem region, and the loop region confirmed the 35 mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.