Dataset Information

Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis.

ABSTRACT: Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (-dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument-even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding.

SUBMITTER: Hu J

PROVIDER: S-EPMC4562029 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Publications

Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis.

Hu Juan J Kim Joonyul J Easley Christopher J CJ

Analytical methods : advancing methods and applications 20150901 17

Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluo ...[more]

PMID: 26366207

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Project description:Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. In this study, we primarily examine the essential hub protein LC8, a small dimer that binds disordered regions of 100+ client proteins in two symmetrical grooves at the dimer interface. Mechanistic details of LC8 binding have remained elusive, hampered in part by ITC data analyses employing simple models that treat bivalent binding as a single event with a single binding affinity. We build on existing Bayesian ITC approaches to quantify thermodynamic parameters for multi-site binding interactions impacted by significant uncertainty in protein concentration. Using a two-site binding model, we identify positive cooperativity with high confidence for LC8 binding to multiple client peptides. In contrast, application of an identical model to the two-site binding between the coiled-coil NudE dimer and the intermediate chain of dynein reveals little evidence of cooperativity. We propose that cooperativity in the LC8 system drives the formation of saturated induced-dimer structures, the functional units of most LC8 complexes. In addition to these system-specific findings, our work advances general ITC analysis in two ways. First, we describe a previously unrecognized mathematical ambiguity in concentrations in standard binding models and clarify how it impacts the precision with which binding parameters are determinable in cases of high uncertainty in analyte concentrations. Second, building on observations in the LC8 system, we develop a system-agnostic heat map of practical parameter identifiability calculated from synthetic data which demonstrates that the ability to determine microscopic binding parameters is strongly dependent on both the parameters themselves and experimental conditions. The work serves as a foundation for determination of multi-step binding interactions, and we outline best practices for Bayesian analysis of ITC experiments.

Dataset Information

Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis.

Publications

Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis.

Similar Datasets

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