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Nucleic Acids Research Advance Access originally published online on April 29, 2008
Nucleic Acids Research 2008 36(11):3522-3530; doi:10.1093/nar/gkn219
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Nucleic Acids Research, 2008, Vol. 36, No. 11 3522-3530
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chemistry

Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity

Joel R. Morgan1, Robert P. Lyon2, Dean Y. Maeda1 and John A. Zebala1,*

1Syntrix Biosystems, Inc., 215 Clay St. NW Suite B-5, Auburn, WA 98001 and 2Seattle Genetics, Inc., 21283 30th Dr SE, Bothell, WA 98021, USA

*To whom correspondence should be addressed. Tel: +1 253 833 8009; Fax: +1 253 833 8127; Email: jzebala{at}syntrixbio.com

Received October 26, 2007. Revised April 8, 2008. Accepted April 9, 2008.

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°C increase in specificity ({Delta}Tm) was observed from snap-to-it probes bearing either terminal iminodiacetic acid ligands coordinated with Ni2+, or terminal dihistidine and nitrilotriacetic acid ligands coordinated with Cu2+. 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.


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