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Nucleic Acids Research, 2002, Vol. 30, No. 22 4952-4959
© 2002 Oxford University Press

Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases

Igor V. Kutyavin, Sergey G. Lokhov, Irina A. Afonina, Robert Dempcy, Alexander A. Gall, Vladimir V. Gorn, Eugene Lukhtanov, Mark Metcalf, Alan Mills, Michael W. Reed, Sylvia Sanders, Irina Shishkina and Nicolaas M. J. Vermeulen*

Epoch Biosciences, 21720 23rd Drive SE, Bothell, WA 98021, USA

*To whom correspondence should be addressed. Tel: +1 425 482 5153; Fax: +1 425 487 1432; Email: nvermeulen{at}epochbio.com

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 (MGBTM 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.


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