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Templating effect in DNA proximity ligation enables use of non-bioorthogonal chemistry in biological fluids.

Artificial DNA, PNA & XNA (2013-02-02)
Nicholas G Spiropulos, Jennifer M Heemstra
ABSTRACT

Here we describe the first example of selective reductive amination in biological fluids using split aptamer proximity ligation (StAPL). Utilizing the cocaine split aptamer, we demonstrate small-molecule-dependent ligation that is dose-dependent over a wide range of target concentrations in buffer, human blood serum and artificial urine medium. We explore the substrate binding preferences of the split aptamer and find that the cinchona alkaloids quinine and quinidine bind to the aptamer with higher affinity than cocaine. This increased affinity leads to improved detection limits for these small-molecule targets. We also demonstrate that linker length and hydrophobicity impact the efficiency of split aptamer ligation. The ability to carry out selective chemical transformations using non-bioorthogonal chemistry in media where competing reactive groups are present highlights the power of the increased effective molarity provided by DNA assembly. Obviating the need for bioorthogonal chemistry would dramatically expand the repertoire of chemical transformations available for use in templated reactions such as proximity ligation assays, in turn enabling the development of novel methods for biomolecule detection.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Benzaldehyde, ReagentPlus®, ≥99%
Sigma-Aldrich
Benzaldehyde, purified by redistillation, ≥99.5%
Supelco
Benzaldehyde, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Quinine, suitable for fluorescence, anhydrous, ≥98.0% (dried material, NT)
Supelco
Benzaldehyde, analytical standard
Sigma-Aldrich
Quinine, 90%
Sigma-Aldrich
Benzaldehyde, natural, FCC, FG
Sigma-Aldrich
Benzaldehyde, puriss. p.a., ≥99.0% (GC)
Sigma-Aldrich
Cinchonidine, 96%
Sigma-Aldrich
Benzaldehyde, ≥98%, FG, FCC
Supelco
Quinine, certified reference material, TraceCERT®
Benzaldehyde, European Pharmacopoeia (EP) Reference Standard