Abstract:
:We describe snap-to-it probes, a novel probe technology to enhance the hybridization specificity of natural and unnatural nucleic acid oligomers using a simple and readily introduced structural motif. Snap-to-it probes were prepared from peptide nucleic acid (PNA) oligomers by modifying each terminus with a coordinating ligand. The two coordinating ligands constrain the probe into a macrocyclic configuration through formation of an intramolecular chelate with a divalent transition metal ion. On hybridization with a DNA target, the intramolecular chelate in the snap-to-it probe dissociates, resulting in the probe 'snapping-to' and binding the target nucleic acid. Thermal transition analysis of snap-to-it probes with complementary and single-mismatch DNA targets revealed that the transition between free and target-bound probe conformations was a reversible equilibrium, and the intramolecular chelate provided a thermodynamic barrier to target binding that resulted in a significant increase in mismatch discrimination. A 4-6 degrees C increase in specificity (DeltaT(m)) was observed from snap-to-it probes bearing either terminal iminodiacetic acid ligands coordinated with Ni(2+), or terminal dihistidine and nitrilotriacetic acid ligands coordinated with Cu(2+). The difference in specificity of the PNA oligomer relative to DNA was more than doubled in snap-to-it probes. Snap-to-it probes labeled with a fluorophore-quencher pair exhibited target-dependent fluorescence enhancement upon binding with target DNA.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Morgan JR,Lyon RP,Maeda DY,Zebala JAdoi
10.1093/nar/gkn219subject
Has Abstractpub_date
2008-06-01 00:00:00pages
3522-30issue
11eissn
0305-1048issn
1362-4962pii
gkn219journal_volume
36pub_type
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