ABSTRACT
A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.
Subject(s)
DNA/chemistry , Oligonucleotide Array Sequence Analysis/methods , RNA/chemistry , Hydrolysis , Indicators and Reagents , Molecular Conformation , Nucleic Acid HybridizationABSTRACT
We show here a new and efficient aqueous chemistry for labeling of any class of nucleic acids for their detection on DNA chip. The labels contain a diazo function as reactive moiety and biotin as detectable unit. The highly selective reaction of diazo group on the phosphate does not disrupt base pairing recognition and hybridization specificity.
Subject(s)
DNA/analysis , DNA/chemistry , Oligonucleotide Array Sequence Analysis/methods , Azo Compounds , Biotinylation , Fluorescent Dyes , Polymerase Chain ReactionABSTRACT
The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines.
Subject(s)
Combinatorial Chemistry Techniques , Nucleic Acid Conformation , Nucleic Acids/chemistry , Base Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, UltravioletABSTRACT
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.
