RESUMO
Various types of minor groove binders have been attached to synthetic oligodeoxynucleotides, and the interactions of these conjugates (MB-ODNs) with DNA are reviewed here. MB-ODNs have enhanced DNA affinity and have improved the hybridization properties of sequence-specific DNA probes. Short MB-ODNs hybridize with ssDNA to give more stable DNA duplexes than unmodified ODNs with similar lengths. Mismatch discrimination of short MB-ODNs is enhanced in comparison to longer unmodified ODNs. The stronger binding of MB-ODNs allows for more stringent hybridization conditions to be used in DNA probe-based assays. MB-ODNs are especially useful in quantitative "real-time" PCR assays since they bind efficiently during the high-temperature primer extension cycle. The synthesis and biophysical chemistry of MB-ODN conjugates are reviewed here. Four published structural classes of MB-ODNs and their various dsDNA binding modes are discussed, and the well-characterized DPI3-type MB-ODNs and their interactions with ssDNA target strands are described in detail.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Composição de Bases/fisiologia , Pareamento Incorreto de Bases , Sítios de Ligação , Bisbenzimidazol/química , Bisbenzimidazol/metabolismo , Catecolaminas/química , Catecolaminas/metabolismo , Ciclopropanos/química , Ciclopropanos/metabolismo , DNA/metabolismo , Sondas de DNA/síntese química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Distamicinas/química , Distamicinas/metabolismo , Corantes Fluorescentes/química , Imidazolinas/química , Imidazolinas/metabolismo , Indóis/química , Indóis/metabolismo , Modelos Biológicos , Modelos Moleculares , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase/métodos , TermodinâmicaRESUMO
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.