Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases.
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.
Kutyavin
Igor V
IV
Epoch Biosciences, 21720 23rd Drive SE, Bothell, WA 98021, USA.
Lokhov
Sergey G
SG
Afonina
Irina A
IA
Dempcy
Robert
R
Gall
Alexander A
AA
Gorn
Vladimir V
VV
Lukhtanov
Eugene
E
Metcalf
Mark
M
Mills
Alan
A
Reed
Michael W
MW
Sanders
Sylvia
S
Shishkina
Irina
I
Vermeulen
Nicolaas M J
NM
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*
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