title: Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine.
abstract: A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Georges
authors.initials: G
authors.lastname: Lahoud
authors.forename: Victor
authors.initials: V
authors.lastname: Timoshchuk
authors.forename: Alexandre
authors.initials: A
authors.lastname: Lebedev
authors.forename: Khalil
authors.initials: K
authors.lastname: Arar
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
authors.forename: Howard
authors.initials: H
authors.lastname: Gamper
mesh_terms: D020029:Base Pairing; D003596:Cytosine / Q000031:analogs & derivatives*; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives*; D015345:Oligonucleotide Probes; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D015345:Oligonucleotide Probes; D014764:Viral Proteins; C024107:2'-deoxycytidine 5'-triphosphate; D006147:Guanine; C029603:deoxyguanosine triphosphate; D003596:Cytosine; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases