Robert Jochem Heetebrij

New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling

Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH. We introduce the principle of COBRA (COmbined Binary RAtio labelling), which is based on the simultaneous use of combinatorial labelling and ratio labelling. Human chromosome painting in 24 colours is accomplished using four fluorophores only. Three fluorophores are used pair wise for ratio labelling of a set of 12 chromosome painting probes. The second set of 12 probes is labelled identically but is also given a binary label (fourth fluorophore). The COBRA method is demonstrated on normal human chromosomes and on a lymphoma (JVM) cell line, using probes enzymatically labelled with fluorescein, lissamine and cy5 as primary fluorophores, and diethylaminocoumarin (DEAC), a blue dye, as combinatorial fourth label to demonstrate incorporated digoxigenin. In addition, the principle was tested using chemical labelling. The first set of 12 painting probes was therefore labelled by ULS (Universal Linkage System), using DEAC, cy3 and cy5 as primary labels, and the second set was labelled similarly, but also contained a digoxigenin-ULS label, which was indirectly stained with fluorescein. Subsequently, a mathematical analysis is presented and methods are indicated for achieving an MFISH multiplicity of 48, 96 or even higher using existing technology.

Platinum(II)‐Based Coordination Compounds as Nucleic Acid Labeling Reagents: Synthesis, Reactivity, and Applications in Hybridization Assays

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono‐ and polynucleotides are described. A [Pt(II)(en)(NH2(CH2)6NH‐tBoc)Cl](NO3) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5′‐monophosphate (5′‐GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic ≈ aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2×10−2 M−1 s−1) and the slowest reacting complex (k=0.93×10−2 M−1 s−1) under these conditions. Platination of an oligodeoxynucleotide (30‐mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.

ULS : a versatile method of labeling nucleic acids for FISH based on a monofunctional reaction of cisplatin derivatives with guanine moieties

The broad extension of an existing chemical DNA labeling technique for molecular cytogenetics is described. Called the Universal Linkage System (ULSTM), it is based on the capability of monoreactive cisplatin derivatives to react at the N7 position of guanine moieties in DNA. Simple repetitive probes, cosmids, PACs, and chromosome-specific painting probes were labeled by ULS and used in a series of multicolor fluorescence in situ hybridization experiments on interphase and metaphase cells. It is demonstrated that ULS-labeled probes, in general, perform as well as the more conventional enzymatically labeled probes. The advantage of ULS labeling over enzymatic labeling techniques is that it is a fast and simple procedure, and that the labeling can easily be scaled up for bulk probe synthesis. In addition, with ULS labeling it is possible to label degraded DNA, a situation in which enzymatic labeling is known to perform unsatisfactorily.

Target Immobilization as a Strategy for NMR-Based Fragment Screening Comparison of TINS, STD, and SPR for Fragment Hit Identification

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM KD is limited. Similarly, they find that SPR detection is most suited to ligands that bind with KD better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.

The new anticancer drug [(2R )-aminomethylpyrrolidine](1,1-cyclobutanedicarboxylato)platinum(II) and its toxic S enantiomer do interact differently with nucleic acids

Abstract The interaction of the two chiral isomers of the new anticancer agent [Pt(ampyr)(cbdca)] (ampyr=aminomethylpyrrolidine, cbdca=cyclobutanedicarboxylate) with 5′-GMP and with short G-containing oligonucleotides has been studied using 1H and 31P NMR, UV-vis spectroscopy and molecular modelling. Each isomer loses the cbdca ligand upon binding to the DNA fragments. Two geometrical isomers of the DNA adducts are formed owing to the presence of the unsymmetric ampyr ligand. These isomers prove to be GG-N7,N7 chelates for d(GpG), d(pGpG) and d(CpGpG). A slight preference for the formation of one geometrical isomer is found in the case of DNA fragments having a phosphate moiety and/or a C base at the 5′-site of the GG sequence. H-bonding interactions from the NH2 moiety towards the 5′-phosphate group and/or the O atom of the C base clearly favour the formation of one geometrical isomer. The presence of these H-bonds, together with the bulky pyrrolidine ring, has resulted in the unique observation (by 1H NMR) of NH protons of coordinated amines that do not rapidly exchange in a 99.95% D2O solution. Temperature-dependence studies show an extremely slow stack ⇄ destack conformational change for the CGG adducts of the S isomer, which could be related to these stable H-bonds of the amine protons towards the oligonucleotide. For the R isomer this stack ⇄ destack conformational change is faster, probably owing to more steric hindrance of the pyrrolidine ring as deduced from the NOESY data, and as also suggested by molecular modelling. The observation of extremely slow rotation around the Pt-N7 bond for [Pt(R-ampyr)(GMP-N7)2] provides further evidence for increased steric hindrance of the R isomer compared to the S isomer. The rate of binding of the drug to G bases proved to be second order for both isomers; in fact the (toxic) S isomer is about two times more reactive than the (non-toxic) R isomer, as seen from k2 values of 0.17±0.01 M–1 s–1 for [Pt(S-ampyr)(cbdca)] and 0.09±0.01 M–1 s–1 for [Pt(R-ampyr)(cbdca)]. No solvent-assisted pathway is involved in these reactions, since the complexes prove to be stable in solution for weeks and therefore only a direct attack of the G base on the Pt must be involved. Because hardly any intermediate species can be detected during the reaction, coordination of the second G base must occur much faster than the binding of the first G base. Since direct attack of the nucleobases takes place, steric interactions become extremely important and therefore are likely to determine the reactivity, activity and even the toxicity of such Pt complexes.

Fluorescence properties, thermal duplex stability, and kinetics of formation of cyanin platinum DNAs.

Fluorescent and haptenized, monofunctionally binding platinum compounds are increasingly used for chemically labeling nucleic acids for hybridization detection purposes. Here we analyze in detail the effect of labeling density of the cyanin-3 and -5 platinum DNA adducts on fluorescence and thermal stability. We also analyzed the kinetics of the reaction of the cyanin platinum compounds with DNA. The data provided are important for the design of optimal platinum DNA labeling and hybridization conditions for fluorescence hybridization applications.

DNA-binding properties of antitumor-active cis-bis(pyridine)platinum(II) organoamides

Abstract The interaction of the new antitumor-active platinum organoamide complexes [Pt{N(p–HC6F4)CH2}2(py)2] and [Pt{N(C6F5)CH2}2(py)2] (py = pyridine) with small G-containing (oligo)nucleotides [GMP, d(GpG)] has been studied to establish whether or not these compounds can bind to DNA in an analogous manner to cisplatin. The reaction products have been analyzed by 1H, 19F and 31P NMR spectroscopy. From the NMR data it is concluded that the {Pt(py)2}2+ moiety binds to the N7 position of the G base, analogously to cisplatin, with the organoamide ligand acting as the leaving group. For the GG-N7,N7 adduct, structural differences are found for the sugar conformation, compared with cisplatin. These differences may account for the activity of these new compounds in tumor cell lines resistant to cisplatin.

A novel approach to site-specifically platinated oligonucleotides applying combinations of nucleobase protecting groups

Platination of a properly protected oligonucleotide followed by deprotection represents a new method for the synthesis of site-specific platinum-modified nucleic acids; N7 platination of 2′-deoxyguanosine can be circumvented by introducing a bulky protective group at the O6 position.