ABSTRACT
A conformational pairing analysis was used to devise nucleobase analogs capable of forming nonselective and energetically favorable base pairs opposite either the purine or the pyrimidine constituents of nucleic acids. 5-methylisocytosine and isoguanine were conceived as a degenerate pyrimidine and a degenerate purine, respectively. Data from previous DNA duplex melting experiments verified that the analogs can act as degenerate nucleobases as hypothesized. Isoguanine also formed unusually stable base pairs with guanine. A quantitative PCR assay yielding equivalent results across hepatitis C virus (HCV) subtypes was created with this system, despite the use of a single probe targeted to a polymorphic region. Amplification curves using probes with 5-methylisocytosine or isoguanine opposite appropriate ambiguous target positions exhibited more signal than curves from similar probes containing common degenerate nucleobase hypoxanthine.
Subject(s)
Nucleic Acid Conformation , Nucleic Acid Hybridization/methods , Nucleotides/chemistry , Base Pairing , DNA Probes , DNA, Viral/analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Hepacivirus/genetics , Hepacivirus/isolation & purification , Nucleic Acid Denaturation , Nucleic Acid Heteroduplexes , Oligonucleotides/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Nucleic acid duplexes associating through purine-purine base pairing have been constructed and characterized in a remarkable demonstration of nucleic acids with mixed sequence and a natural backbone in an alternative duplex structure. The antiparallel deoxyribose all-purine duplexes associate specifically through Watson-Crick pairing, violating the nucleobase size-complementarity pairing convention found in Nature. Sequence-specific recognition displayed by these structures makes the duplexes suitable, in principle, for information storage and replication fundamental to molecular evolution in all living organisms. All-purine duplexes can be formed through association of purines found in natural ribonucleosides. Key to the formation of these duplexes is the N(3)-H tautomer of isoguanine, preferred in the duplex, but not in aqueous solution. The duplexes have relevance to evolution of the modern genetic code and can be used for molecular recognition of natural nucleic acids.
