Invader probes: harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications

Development of probes capable of recognizing specific regions of chromosomal DNA has been a longstanding goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced doublestranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Herein, probes with different chemistries and architectures – varying in the position, number, and distance between the intercalator zippers – are studied with respect to hybridization energetics and DNAtargeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 mM), fast (t50 < 3 h), kinetically stable (>24 h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in nondenaturing fluorescence in situ hybridization experiments for detection of gender-specific mixedsequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.