Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity.
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.
Morgan
Joel R
JR
Syntrix Biosystems, Inc., 215 Clay St. NW Suite B-5, Auburn, WA 98001, USA.
Lyon
Robert P
RP
Maeda
Dean Y
DY
Zebala
John A
JA
D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes; D008670:Metals / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D015341:Nucleic Acid Probes / Q000737:chemistry*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D013696:Temperature
D005456:Fluorescent Dyes; D008670:Metals; D015341:Nucleic Acid Probes; D020135:Peptide Nucleic Acids; D004247:DNA