Pyrene-functionalized oligonucleotides (PFOs) are increasingly explored as tools in fundamental research, diagnostics and nanotechnology. Their popularity is linked to the ability of pyrenes to function as polarity-sensitive and quenchable fluorophores, excimer-generating units, aromatic stacking moieties and nucleic acid duplex intercalators. These characteristics have enabled development of PFOs for detection of complementary DNA/RNA targets, discrimination of single nucleotide polymorphisms (SNPs), and generation of π-arrays on nucleic acid scaffolds. This critical review will highlight the physical properties and applications of PFOs that are likely to provide high degree of positional control of the chromophore in nucleic acid complexes. Particular emphasis will be placed on pyrene-functionalized Locked Nucleic Acids (LNAs) since these materials display interesting properties such as fluorescence quantum yields approaching unity and recognition of mixed-sequence double stranded DNA (144 references).

Very efficient interstrand communication systems in nucleic acid duplexes, based on pyrene excimer formation between 2′-N-(pyren-1-yl)methyl-2′-amino-LNA monomers, demonstrate the versatility of functionalized 2′-amino-LNA monomers for Ångström-scale chemical engineering.

A lateral flow test strip assay, enabling sensitive detection of DNA specific to the foodborne pathogen E. coli O157:H7, is described. The use of LNA-conjugated gold nanoparticle probes, along with signal amplification protocols, results in minimum detectable concentrations of ∼0.4 nM.

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. Recognition of dsDNA 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.