Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.

In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).

DNA bis-intercalators as new anti-tumour agents: modulation of the anti-tumour activity by the linking chain rigidity in the ditercalinium series.

Ditercalinium (2,2'-([4,4'-bipiperidine-1,1'-diyl] di-2,1-ethane diyl) bis (10-methoxy-7H-pyrido[4,3c] carbazolium) tetra(methyl sulphonate--NSC 366241), a DNA bis-intercalating compound presently under clinical trial, elicits an original mechanism of action and thus appears as the first of a new class of anti-tumour drugs. Previous studies have shown that a reduced flexibility of the linking chain of these dimers is essential for their biological activity. In order to analyze their mechanism of action at the molecular level and to obtain structure-activity relationships in this series, new derivatives with additional methylene groups between the two piperidine rings have been synthesized. Whereas the addition of a single methylene group in the chain preserves the anti-tumour activity of the dimers, the addition of a second methylene diminishes it; the addition of three methylenes completely abolishes it. Lengthening of sonicated DNA and unwinding of supercoiled DNA support a bis-intercalation mechanism for these drugs. In addition, analyses of poly d(A-T) melting curves in the presence of the drugs, and competition experiments with ethidium dimer, show that these compounds bind to DNA with high affinity (10(7)-10(8) M-1). N.m.r. studies of the dimers in aqueous medium show that the introduction of a single methylene group in the linker leads to compounds with a conformationally-induced decrease of intermolecular stacking interactions, which might be related to the DNA affinity enhancement observed for these dimers. Different hypotheses concerning structure-activity relationships in the different series are discussed.

Rational design of bis-intercalating drugs as antitumour agents: importance of rigidity in the linking chain.

Ditercalinium (NSC 366241), a dimer of 10-methoxy-7H-pyrido[4,3-c]carbazole quaternarized on the pyridine nitrogen by a rigid bis(1,1'-ethyl)-4,4'-bipiperidine linking chain, is endowed with antitumour properties and bis-intercalates with high affinity into DNA. New dimers have been designed in the same series to evaluate the importance of the rigidity of the linking chain for pharmacological activity. The dimers, characterized by one and two additional methylene groups between the two piperidine rings of the linking chain, remain as active as ditercalinium. However, a third additional CH2 group between the two piperidine rings leads to an inactive dimer. Relationships between the different pharmacological activities of the drugs and their intercalation complexes with DNA were investigated using viscosimetry, absorption spectroscopy and NMR analyses.