Triplex nucleic acids have attracted attention due to their potential regulation of DNA transcription, replication, recombination, chromosome packing, and potential applications in gene therapy.
The ‘canonical triplex’ structures of these studies have consisted of long stretches of uninterrupted homopyrimidine or homopurine strands, typically 12–14 bases in length, that bind in the major groove of duplex DNA, forming Hoogsteen or reverse Hoogsteen hydrogen bonds with bases in the homopurine or purine-rich strand of the duplex.
Substantial deviations from strict binding rules are understood to preclude triplex formation. The utility of such ‘canonical triplexes’ is severely limited due to the requirement of long uninterrupted homopurine sequences in the double-stranded DNA (dsDNA). Such sequence requirements have been understood to preclude the development of triplex-based molecular diagnostics, which is why human genomic samples have, up to now, been considered difficult to assay directly without denaturation or PCR amplification.
However, a team of scientists based in Ontario, Canada, has reported the use of a base-specific heteropolymeric triplex to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA. Formed by a native duplex genomic target and an oligonucleotide third strand probe, the triplex can also assay human genomic duplex DNA for single nucleotide polymorphisms (SNP), without PCR amplification.
Writing in the international, peer-reviewed, open access, online journal, PLoS ONE(*), Jasmine Daksis and Glen Erikson from Mississauga-based Ingeneus Research describe how they used wild-type and mutant probes to identify triplexes containing FVLG1691A, MTHFRC677T and CFTR mutations.
The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex.
The bases of the stable duplex target are rendered reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo.
The authors note that their assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are ‘canonical triplexes’. Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. “The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays,” they write.
In the article they present a wealth of data relating to the assay of pathogens in samples also containing human genomic duplex DNA and to the assay of SNPs present in human genomic samples. Reactions can be monitored after as little as five minutes. The highly sensitive diagnostic assay allows for the direct detection of base sequence in human genomic duplex samples, thereby obviating the use of PCR with its inherent problems and cost.
YOYO-1 de-condenses the duplex target, which renders the duplex nucleic acid readily reactive to oligo ssDNA probes. Any sequence present in the duplex may be assayed. It is surmised that specific third strand binding creates additional grooves into which additional YOYO-1 molecules intercalate.
Their Genome Flow instrument (Fig.1) employs hardware from FIALab Instruments of Bellevue, Washington. The company specialises in flow injection analysis (FIA), an analytical technique based on microfluidic manipulation of samples and reagents. Samples are injected into a carrier/reagent solution that transports the sample zone into a detector while the desired chemical/biochemical reactions take place.
The instrument used by Daksis has one moving part, the syringe pump. It allows samples to be automatically quantified, a necessary step in the genomic assay because samples must be brought to a standard concentration so they can be mixed with standard amounts of oligo probes for the purpose of automatic in solution assay.
