title: Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.
abstract: 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)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (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 approximately aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2 x 10(-2) M(-1) s(-1)) and the slowest reacting complex (k=0.93 x 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.
authors.affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. heetebri@chem.leidenuniv.nl
authors.forename: R J
authors.initials: RJ
authors.lastname: Heetebrij
authors.forename: E G
authors.initials: EG
authors.lastname: Talman
authors.forename: M A
authors.initials: MA
authors.lastname: v Velzen
authors.forename: R P M
authors.initials: RP
authors.lastname: van Gijlswijk
authors.forename: S S
authors.initials: SS
authors.lastname: Snoeijers
authors.forename: M
authors.initials: M
authors.lastname: Schalk
authors.forename: J
authors.initials: J
authors.lastname: Wiegant
authors.forename: F
authors.initials: F
authors.lastname: v d Rijke
authors.forename: R M
authors.initials: RM
authors.lastname: Kerkhoven
authors.forename: A K
authors.initials: AK
authors.lastname: Raap
authors.forename: H J
authors.initials: HJ
authors.lastname: Tanke
authors.forename: J
authors.initials: J
authors.lastname: Reedijk
authors.forename: H-J
authors.initials: HJ
authors.lastname: Houthoff
mesh_terms: D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D005399:Fishes; D020869:Gene Expression Profiling / Q000379:methods; D006157:Guanosine Monophosphate / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007700:Kinetics; D008297:Male; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D015341:Nucleic Acid Probes / Q000138:chemical synthesis / Q000737:chemistry*; D009696:Nucleic Acids / Q000032:analysis* / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009944:Organoplatinum Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D013054:Spectrophotometry, Atomic; D013094:Spermatozoa / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D015341:Nucleic Acid Probes; D009696:Nucleic Acids; D009838:Oligodeoxyribonucleotides; D009944:Organoplatinum Compounds; D012313:RNA; D006157:Guanosine Monophosphate; D004247:DNA