Invader LNA: Efficient targeting of short double stranded DNA

Abstract

Despite progress with triplex-forming oligonucleotides or helix-invading peptide nucleic acids (PNAs), there remains a need for probes facilitating sequence-unrestricted targeting of double stranded DNA (dsDNA) at physiologically relevant conditions. Invader LNA probes, i.e., DNA duplexes with "+1 interstrand zipper arrangements" of intercalator-functionalized 2'-amino-α-L-LNA monomers, are demonstrated herein to recognize short mixed sequence dsDNA targets. This approach, like pseudo-complementary PNA (pcPNA), relies on relative differences in stability between probe duplexes and the corresponding probe:target duplexes for generation of a favourable thermodynamic gradient. Unlike pcPNA, Invader LNA probes take advantage of the "nearest neighbour exclusion principle", i.e., intercalating units of Invader LNA monomers are poorly accommodated in probe duplexes but extraordinarily well tolerated in probe-target duplexes (ΔT m /modification up to +11.5 °C). Recognition of isosequential dsDNA-targets occurs: a) at experimental temperatures much lower than the thermal denaturation temperatures (T m 's) of Invader LNAs or dsDNA-targets, b) at a wide range of ionic strengths, and c) with good mismatch discrimination. dsDNA recognition is monitored in real-time using inherent pyrene-pyrene excimer signals of Invader LNA probes, which provides insights into reaction kinetics and enables rational design of probes. These properties render Invader LNAs as promising probes for biomedical applications entailing sequence-unrestricted recognition of dsDNA.

Synthesis and characterization of oligodeoxyribonucleotides modified with 2′-thio-2′-deoxy-2′-S-(pyren-1-yl)methyluridine

“…Consistent with our previous observations with other Invader chemistries, 6,7,12,14,15 duplexes with +1 interstrand zippers of S monomers are less stable than probes with other zipper arrangements (compare T m 's for S2:S5 relative to other probe duplexes, Table 4). The energetically activated nature of S2:S5 was verified through analysis of thermodynamic parameters, which were obtained from denaturation curves.…”

“…6,12-15 In the present work, we set out to study the influence of the 2′-heteroatom of the pyrene-functionalized nucleotide monomer on the dsDNA-recognition characteristics of Invader probes. We hypothesized that the lower electronegativity of the sulfur atom of 2′- S -(pyren-1-yl)methyl-2′-thiouridine monomer S would weaken the gauche effect between O4′ and the 2′-substituent, and thus increase the population of C2′- endo ( South -type) furanose conformations.…”

“…6,13-15 We therefore selected to evaluate the dsDNA-recognition efficiency of S2:S5 using a 3′-digoxigenin (DIG) labeled DNA hairpin (DH) as a model dsDNA target, which is comprised of a 9-mer double-stranded mixed sequence stem linked by a T 10 loop (Figure 4). However, incubation of DH1 with S2:S5 in a HEPES buffer for 12-16 hours at ambient temperatures did not result in formation of slower-migrating recognition complexes on non-denaturing PAGE gels even at 500-fold molar probe excess (Figure 4).…”

Recognition of mixed-sequence DNA using double-stranded probes with interstrand zipper arrangements of O2′-triphenylene- and coronene-functionalized RNA monomers

“…25 Subsequent studies revealed that the synthetically more readily available 2′- O -(pyren-1-yl)methyl-RNA monomers can be used instead with minimal or no loss in dsDNA-recognition efficiency. 20 In fact, we have shown that Invader probes based on the 2′- O -(pyren-1-yl)methyl-RNA chemistry can be used for recognition of mixed-sequence chromosomal DNA targets at non-denaturing conditions.…”

Sandwich assay for mixed-sequence recognition of double-stranded DNA: Invader-based detection of targets specific to foodborne pathogens

“…1 bottom). 16-22 We have previously used the stability difference between Invaders and probe-target duplexes to realize mixed-sequence recognition of linear dsDNA targets, 16,17 DNA hairpins 18,19 and chromosomal DNA. 18 …”