A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

Abstract

Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers with high target specificity and affinity, and the ability to undergo the required conformational changes. Here we report on an alternative generic scheme for detecting small molecules and proteins in solution based on a shift in the equilibrium of DNA-based strand displacement competition reaction. The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies or protein targets of these molecules. The detection scheme provides a generic alternative to aptamers for detection of analytes.

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

“…It is known that the T m of a duplex depends on the tag on the DNA strand in the duplex (33). Therefore, an internal biotin tag was used to decrease the T m of the hybrid formed between the template and target sequence.…”

Materiomics for Oral Disease Diagnostics and Personal Health Monitoring: Designer Biomaterials for the Next Generation Biomarkers

“…DNA sensors have been used to detect DNA (Rogers et al, 2001;Wang et al, 1998), proteins (Ban et al, 2004;Leung et al, 2012), and even small molecules/ions (Li and Lu, 2000;Zhang et al, 2014) in the form of optical Rogers et al, 2001;Vo-Dinh et al, 2001), electrochemical (Kinsella and Ivanisevic, 2007;Odenthal and Gooding, 2007;Wang, 2006), or mass-sensitive (Garcia-Martinez et al, 2011;Mannelli et al, 2005) for a variety of biological applications. One particular example can be using DNA probes to differentiate healthy, gingivitis, and periodontitis subjects using DNA-DNA hybridization techniques (Palys et al, 1998).…”

Novel selection methods for DNA-encoded chemical libraries

“…In the strand-displacement competition assay developed by Gothelf and coworkers [51], a DNA-linked small molecule is hybridized with a complementary oligonucleotide. Protein-small molecule binding decreases the affinity of this duplex, enabling toehold displacement of the complementary strand.…”