DNA as a Force Sensor in an Aptamer-Based Biochip for Adenosine

Abstract

Without prior signal amplification, small molecules are difficult to detect by current label-free biochip approaches. In the present study, we developed a label-free capture biochip based on the comparative measurement of unbinding forces allowing for direct detection of small-molecule-aptamer interactions. The principle of this assay relies on increased unbinding forces of bipartite aptamers due to complex formation with their cognate ligands. The bipartite aptamers are immobilized on glass support via short DNA duplexes that serve as references to which unbinding forces can be compared. In a simple model system, adenosine is captured from solution by an adenosine-selective aptamer. Linking the molecular chains, each consisting of a short DNA reference duplex and a bipartite aptamer, between glass and a poly(dimethylsiloxane) (PDMS) surface and subsequently separating the surfaces compares the unbinding forces of the two bonds directly. Fluorescence readout allows for quantification of the fractions of broken aptamer and broken reference bonds. The presence of micromolar adenosine concentrations reliably resulted in a shift toward larger fractions of broken reference bonds. Because of the force-based design, the interactions between the bipartite aptamer and the target, rather than the presence of the target, are detected and no washing step disturbing the equilibrium state prior to probing and no reporter aptamer or antibody is required. The assay exhibits excellent selectivity against other nucleotides and detects adenosine in the presence of a complex molecular background. Multiplexing was demonstrated by performing whole titration experiments on a single chip revealing an effective half-maximal concentration of 124.8 microM agreeing well with literature values.

Adhesion through Single Peptide Aptamers

“…Aptamers 1 and antibodies are two broad categories of biomolecules with specific binding affinity, enabling applications in sensing, 2 diagnostic, 3 drug delivery, 4 imaging 5 and therapy. 6,7 Peptide aptamers typically contain 8–20 amino acids and bind materials or biomolecules.…”

Optical Thermophoresis for Quantifying the Buffer Dependence of Aptamer Binding

“…[5] Aptamers have been implemented in a variety of sensing technologies [6] including optical approaches like "aptamer beacons", [7] electronic-sensing strategies, [8] and techniques based on changes in mass [9] or force. [10] In most aptamer-based binding assays, the signal transduction mechanism depends on the molecular recognition mechanism. As a result the aptamers must be designed not only to adopt an appropriate conformation to bind to a target (recognition) but also to undergo a binding-induced conformational change, which affects the fluorescence of a dye [8] or the electron transfer [9] of a redox tag to an electrode (signal transduction).…”

A high throughput molecular force assay for protein–DNA interactions

“…For this, it follows that the MFA is capable of screening for protein-DNA interactions comparable to PBMs, ChIP-chip and MITOMI. Compared to these high-throughput methods the following advantages arise: (i) no stringent washing between force measurement and readout is needed, (ii) a wide range of affinities is accessible, even weak binders, 26 (iii) a quantitative and robust analysis due to the simple image division for normalization, (iv) and no label or marker against the protein is needed since the MFA relies on the detection of the specific interaction of binding protein and DNA.…”