DNA destabilization

Florian, J. Goodman, M.F. Warshel, A., Free energy perturbation calculations of DNA destabilization by base substitutions the effect of neutral guanine-thymine, adenine-cytosine and adenine-difluorotoluene mismatches, J. Phys. Chem. B 2000,104, 10092-10099. 

This work provides important evidence for elucidating the cytotoxic effect of the ruthenium-arene complexes and the influence of the arene thereon, for instance with respect to excision repair of DNA lesions and DNA destabilization. It also established two different classes of Ru(II) arene anticancer drugs, i.e. those bearing an arene that has the possibility to intercalate and those that do not. This distinction is important as we will see further differences in DNA binding interactions for these two classes (vide infra). 

By accepting the plausibility of satisfying these requlrsments a number of predictive features csn be seen about the cmiditlon and elements of DNA destabilization vivo and vitro, which account for many general observations described in the literature. These are listed as "Predictions. ... 

The nature of DSBs directly implies that processing mechanisms need to solve the problem of DNA destabilization caused by the disruption in the continuity of the molecule, the problem of sequence restoration near the DSB, and possibly also the problem generated by the presence of other forms of DNA damage near the break. The information summarized in the following sections indicates that despite these extra requirements, cells are... 

Thirty percent of the tumor-derived mutations are in L3, which contains the single most frequently mutated residue, Arg 248. Clearly the interaction between DNA and the specific side chain of an arginine residue inside the minor groove is of crucial importance for the proper function of p53. It is an open question whether this interaction is needed for the recognition of specific DNA sequences, or is required for the proper distortion of the DNA structure, or a combination of both. Other residues that are frequently mutated in this region participate in interactions with loop L2 and stabilize the structures of loops L2 and L3. Mutations of these residues presumably destabilize the structure so that efficient DNA binding can no longer take place. 

Figure 1. The cell cycle as a Cdc2 cycle. Progression through the eukaryotic cell cycle is sensitive to the phosphorylation state of Cdc2. A block to DNA synthesis (S) prevents dephosphorylation, and hence activation, of Cdc2. Impaired spindle function will prevent deactivation of Cdc2 and thus blocks exit from M phase (Hoyt et al., 1991 Li and Murray, 1991 reviewed in Nurse, 1991). Exit from M phase requires destruction of the regulatory subunit, Cyc B. Dephosphorylation of Cdc2 at thr-161 may act to destabilize the Cdc2/Cyc B complex and thus allow the ubiquitination of Cyc B followed by its destruction.

Further experiments focused therefore on [RuCl(en)(r 6-tha)]+ (12) and [RuCl(rj6-p-cym)(en)]+ (22), which represent the two different classes, and their conformational distortion of short oligonucleotide duplexes. Chemical probes demonstrated that the induced distortion extended over at least seven base pairs for [RuCl(rj6-p-cym)(en)]+ (22), whereas the distortion was less extensive for [RuCl(en)(rj6-tha)]+ (12). Isothermal titration calorimetry also showed that the thermodynamic destabilization of the duplex was more pronounced for [RuCl(r 6-p-cym)(en)]+ (22) (89). DNA polymerization was markedly more strongly inhibited by the monofunctional Ru(II) adducts than by monofunctional Pt(II) compounds. The lack of recognition of the DNA monofunctional adducts by HMGB1, an interaction that shields cisplatin-DNA adducts from repair, points to a different mechanism of antitumor activity for the ruthenium-arenes. DNA repair activity by a repair-proficient HeLa cell-free extract (CFE) showed a considerably lower level of damage-induced DNA repair synthesis (about six times) for [RuCl(en)(rj6-tha)] + compared to cisplatin. This enhanced persistence of the adduct is consistent with the higher cytotoxicity of this compound (89). 

The simultaneous mutation of 2-imidazole-distamycin to distamycin at both the sites I and II led to a free energy change of -1.8 kcal/mol (AGg - 2 AG3). The NMR experiments showed that the relative populations of Dst Dst DNA and 2-ImD 2-ImD DNA are 50 1 giving an experimental free energy difference of -2.3 kcal/mol (AG7 - AG2). This indicates that the favorable van der Waals interactions between distamycin and DNA at sites I and II and the stacking interactions between the two distamycin molecules stabilize the 2 1 Dst DNA complex over the 2 1 2-ImD DNA complex. The major destabilization factor for the 2 1 2-ImD DNA complex is the lack of... 

Release of DNA in vivo takes place due to the increased acidic conditions inside living cells that result in the destabilization of the ORMOSIL-DNA complex. SiCVbased nanoparticles, in fact, do not release encapsulated biomolecules because of the strong hydrogen bonding between the biomolecule s polar centres and the silanols at the cage surface (as ORMOSIL-entrapped hydrophobic molecules are not leached in aqueous systems due to strong hydrophobic interactions).17... 

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

With the aim to reduce unspecific protein interactions while controlling DNA release, acid-sensitive PEG has been designed. Several pH-sensitive systems are described in Volume I, Chapter 8. Briefly, the objective is to develop molecules for biological purposes, which means molecules able to be destabilized at slight pH changes. Tumors or ischemia sites present... 

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