The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w-and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c-and y-ions. (J Am Soc Mass Spectrom 2002, 13, 936 -945)
The first step of the reaction between DNA and the antitumor drug cisplatin or its clinically inactive isomer transplatin yields monofunctional adducts. Most of the cisplatin monofunctional adducts further react and rather rapidly (t(1/2) smaller than a few hours) to form intrastrand and interstrand cross-links. It is generally accepted that the clinical activity of cisplatin is related to the formation of bifunctional lesions. As concerns transplatin, several studies disagree on the rate of closure of the monofunctional adducts and the nature of the bifunctional lesions. In order to explain these discrepancies, we have prepared several duplexes containing a single monofunctional trans-[Pt(NH3)2(dG)Cl]+ adduct and zero to two monofunctional [Pt(dien)(dG)]2+ adducts at defined positions. In these duplexes, the inert [Pt(dien)(dG)]2+ adducts mimic the presence of transplatin monofunctional adducts. We show that the closure of the transplatin monofunctional adducts is strongly affected by the presence of other adducts and by the length of the duplexes. These findings suggest that the discrepancies in the literature originate from the nature of the platinated samples (molar ratio of bound platinum per nucleotide, length of the DNA fragments). Our general conclusion is that within transplatin-modified DNA, at a low level of platination, the monofunctional adducts evolve slowly (t(1/2) > 24 h) into bifunctional lesions and that these bifunctional lesions are mainly interstrand cross-links. This could explain, at least in part, the clinical inefficiency of transplatin.
We previously introduced a method called steroid-mediated gene delivery (SMGD), which uses steroid receptors as shuttles to facilitate the nuclear uptake of transfected DNA. Here, we describe a SMGD strategy with peptide nucleic acids (PNAs) that allowed linkage of a steroid molecule to a defined position in a plasmid without disturbing its gene expression. We synthesized and tested several bifunctional steroid derivatives [patent in process of nationalization] and finally selected the compound named DEX-bisPNA, a molecule consisting of a dexamethasone moiety linked to a PNA clamp (bisPNA) through a 30-atom chemical spacer. Dex-bisPNA binds to the glucocorticoid receptor (GR) as well as to reporter plasmids containing the corresponding PNA binding sites, translocates the GR from the cytoplasm into the nucleus, and increases the delivery of plasmid to the nucleus, resulting in enhanced GR-dependent expression of the reporter gene. The SMGD effect was more pronounced in growth-arrested cells than in proliferating cells. The specificity for the GR was shown by the reversion of the SMGD effect in the presence of dexamethasone as well as an enhanced expression in GR-positive cells but not in GR-negative cells. Thus, SMGD with PNA is a promising strategy for nonviral gene delivery into target tissues expressing specific steroid receptors.
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