Recognition of double-stranded DNA using LNA-modified toehold Invader probes

Abstract

Development of molecules capable of binding to specific sequences of double-stranded (ds) DNA continues to attract considerable interest, as this can yield useful tools for applications in life science, biotechnology,...

Nicked Invader probes: multistranded and sequence-unrestricted recognition of double-stranded DNA

“…As previously reported for conventional and toehold Invader probes, 25,40 duplexes between individual probe strands and ssDNA harboring complementary target regions are much more stable than the corresponding double-stranded probe segments ( e.g. , compare T m = 73.0 °C, 74.5 °C, and 37.0 °C, for ON5 : DNA2 , ON8 : DNA1 and ON5 : ON8 , respectively).…”

“…S26†).cData previously reported in ref. 40.dSee discussion of Fig. S26 and S27 in ESI.†eDifferential denaturation experiments confirm this transition (Fig.…”

“…In fact, binding is even more specific than what we previously observed with toehold Invader probes. 40 Presumably, the high binding specificity is the consequence of multiple effects including (i) stringency clamping effects, i.e. , greater stability differences between matched and mismatched recognition complexes due to the metastable nature of the structured probes, 44 (ii) avoidance of forming multiple, energetically unfavorable, double-stranded segments containing mismatched base-pairs, 45 and (iii) high binding specificity of short oligonucleotides.…”

“…1b). 40 The advantage of toehold Invader probes vis-à-vis the corresponding blunt-ended Invader probes is that longer dsDNA regions can be targeted since the double-stranded region of the probe can be shortened, thus facilitating dissociation. Moreover, affinity-enhancing modifications such as LNA 41 monomers can be incorporated in the single-stranded overhangs to further increase cDNA affinity.…”

“…Toehold Invader probes have been shown to result in more efficient recognition of complementary dsDNA regions, allowing for recognition of chromosomal DNA targets that were refractory to recognition by conventional blunt-ended Invader probes. 40…”