Oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. Here we introduce bivalent peptide-DNA conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toehold-mediated strand displacement reactions. Employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nM concentrations of peptide-DNA locks and oligonucleotide displacer strands. Introduction of two different toehold strands on the peptide-DNA lock allowed signal integration of two different inputs, yielding logic OR- and AND-gates. The range of molecular inputs could be further extended to protein-based triggers by using protein-binding aptamers.

Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1 : 1 antibody-ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide-dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide-dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals.

DNA-templated reversible assembly of an enzyme-inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-β-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template strand. Formation of a rigid dsDNA linker upon addition of a complementary target strand disrupts the enzyme-inhibitor complex and results in the restoration of enzyme activity, enabling detection of as little as 2 fmol DNA. The noncovalent assembly of the complex allows easy tuning of target and template strands without changing the oligonucleotide-functionalized enzyme and inhibitor domains. Using a panel of eight different template sequences, restoration of enzyme activity was only observed in the presence of the target viral DNA sequence. The use of stable, well-characterized protein domains and the intrinsic modularity of our system should allow easy integration with DNA/RNA-based logic circuits for applications in biomedicine and molecular diagnostics.

We previously reported the development of high affinity Zn(2+) FRET sensors based on the Zn(2+)-mediated interaction between the CXXC motifs present in the copper chaperone proteins ATOX1 and WD4. By systematically substituting several of these cysteines for methionines, we constructed sensor variants that retain a high affinity for Cu(+), while effectively abolishing their ability to form stable tetrahedral Zn(2+) complexes.

This feature article discusses the development of generic strategies to dynamically control protein activity via DNA-based triggers.