title: Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases.
abstract: Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson-Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson-Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB(TM) is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5'-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.
authors.affiliation: Epoch Biosciences, 21720 23rd Drive SE, Bothell, WA 98021, USA.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Kutyavin
authors.forename: Sergey G
authors.initials: SG
authors.lastname: Lokhov
authors.forename: Irina A
authors.initials: IA
authors.lastname: Afonina
authors.forename: Robert
authors.initials: R
authors.lastname: Dempcy
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Gall
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Gorn
authors.forename: Eugene
authors.initials: E
authors.lastname: Lukhtanov
authors.forename: Mark
authors.initials: M
authors.lastname: Metcalf
authors.forename: Alan
authors.initials: A
authors.lastname: Mills
authors.forename: Michael W
authors.initials: MW
authors.lastname: Reed
authors.forename: Sylvia
authors.initials: S
authors.lastname: Sanders
authors.forename: Irina
authors.initials: I
authors.lastname: Shishkina
authors.forename: Nicolaas M J
authors.initials: NM
authors.lastname: Vermeulen
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D005090:Exodeoxyribonucleases; D005453:Fluorescence; D006147:Guanine / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009705:Nucleosides / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011720:Pyrazoles / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D011744:Pyrimidinones / Q000737:chemistry*
chemical_list: C468765:8-aza-7-deazaguanine; D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes; D009705:Nucleosides; D009838:Oligodeoxyribonucleotides; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; C014175:pyrazolo(3,4-d)pyrimidine; D006147:Guanine; D005090:Exodeoxyribonucleases