Abstract
DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody–epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.
Highlights
signalto-background ratio (S/B) ratio Time (h)T length (No of nt) translator module, showing a maximal S/B ratio of B20 using a toehold of 3 nucleotides.ordinary differential equations (ODEs) model of the antibody-templated strand exchange (ATSE) module
We have shown that Antibody-Templated Strand Exchange (ATSE) of peptide-functionalized DNA strands provides a unique and robust molecular approach to translate the presence of an antibody into a ssDNA output
Both thermodynamic and kinetic effects contribute to the remarkable efficiency of ATSE
Summary
S/B ratio Time (h)T length (No of nt) translator module, showing a maximal S/B ratio of B20 using a toehold of 3 nucleotides.ODE model of the ATSE module. Having established the kinetic parameters kf, kbg and krep and the Kd for the antibody–epitope interaction allowed the determination of the rate constants (kintra) for the antibodytemplated intramolecular toehold-mediated strand exchange reaction as a function of toehold lenght by non-linear least square optimization of the ODE model to experimental data obtained in the presence of 2 nM anti-HA antibody (Fig. 2c; Supplementary Table 6). Using the experimentally obtained kinetic parameters kb, kf, krep, kbg and kintra, we simulated the concentration of the individual reaction components as a function of time for the system with the optimal toehold length of 3 nucleotides (Fig. 2d; Supplementary Fig. 19) This simulation shows that the amount of free output O increases in the first few minutes of the reaction, indicating that the anti-HA antibody (Ab) rapidly induces the toehold-mediated strand exchange reaction to form the intramolecular cyclic complex
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