Toehold probes utilize the thermodynamic properties of specifically designed oligonucleotides for specific detection of a target sequence. Correctly designed toehold probes selectively detect the correct target and prevent the non-specific detection of target sequences that differ by a single non-matching nucleotide.
DNA oligonucleotides containing bridged nucleic acids (BNAs) or locked nucleic acids (LNAs) are known to bind selectively to target DNA at room temperature. These oligonucleotides include a toehold sequence binding at the end of the target allowing for selective enrichment and purification of DNA fragments. Force spectroscopy revealed that probe-target hybrids are sufficiently stable to withstand forces over 65 pN allowing determination of force-extension curves. However, the incorporating of BNA nucleotides into toehold probes has limitations since this is sequence dependent thereby limiting the number of BNAs per oligonucleotide probe sequence. Also, depending on the sequence, oligonucleotides with a high BNA content are prone to self-hybridization. Therefore the trend for self-hybridization of the oligonucleotides will need to be checked with an oligonucleotide design tool found on several websites.
Reference Toeholds: https://www.ncbi.nlm.nih.gov/pmc/?term=toehold Hermans, Nicolaas; Huisman, Juriën Jori; Brouwer, Thomas Bauke; Schächner, Christopher; van Heusden, G. Paul H.; Griesenbeck, Joachim; van Noort, John; Toehold-enhanced LNA probes for selective pull down and single-molecule analysis of native chromatin. (2017) Scientific Reports 7(1) 16721. https://www.nature.com/articles/s41598-017-16864-7
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