Antibody-powered nucleic acid release using a DNA-based nanomachine

Abstract

A wide range of molecular devices with nanoscale dimensions have been recently designed to perform a variety of functions in response to specific molecular inputs. Only limited examples, however, utilize antibodies as regulatory inputs. In response to this, here we report the rational design of a modular DNA-based nanomachine that can reversibly load and release a molecular cargo on binding to a specific antibody. We show here that, by using three different antigens (including one relevant to HIV), it is possible to design different DNA nanomachines regulated by their targeting antibody in a rapid, versatile and highly specific manner. The antibody-powered DNA nanomachines we have developed here may thus be useful in applications like controlled drug-release, point-of-care diagnostics and in vivo imaging.

Figure 1. Working principle of antibody-powered DNA-based nanomachine.

A DNA strand (black) labelled with two antigens (green hexagons) can load a nucleic acid strand (blue) through a clamp-like triplex-forming mechanism. The binding of a bivalent macromolecule (here an antibody) to the two antigens causes a conformational change that reduces the stability of the triplex complex with the consequent release of the loaded strand.

Figure 2. Designing the antibody-powered nanomachine.

To find the optimal DNA cargo length to observe the antibody-induced release from the nanomachine, we have compared the binding affinity of a triplex-forming nanomachine with that of a control nanomachine able to only form a duplex complex (a) using cargo strands of different length (13 nt (b), 12 nt (c), 11 nt (d) and 10 nt (e)). We have observed the strongest difference in affinity (here depicted as the difference of the relative occupancy) between the triplex-forming nanomachine and the control nanomachine with the 12-nt DNA cargo (f,g). (h,i) Using the 12-nt DNA cargo, we have also performed melting denaturation experiments showing that, while the triplex complex is stable up to 50 °C (T_m=52.1±0.5 °C), the nanomachine/cargo complex solely based on Watson–Crick interactions (control) shows a melting temperature of 37.0±0.5 °C. The experiments in this figure were performed using a DNA nanomachine (either triplex-forming or control) labelled with a fluorophore/quencher pair (FAM and BHQ-1) so that the binding of the DNA cargo can be easily followed through the decrease or increase, respectively, of the fluorescence signal. The binding curve experiments were performed in 50 mM Na₂HPO₄, 150 mM NaCl and 10 mM MgCl₂ at pH 6.8, 37 °C at a concentration of nanomachine of 3 nM and adding increasing concentrations of cargo strand. Melting curve experiments were performed using the same buffer solution at an equimolar concentration (10 nM) of nanomachine and 12-nt cargo.

Figure 3. Antibody-powered DNA-based nanomachine.

(a) We first used digoxigenin (Dig) as antigen and anti-Dig antibodies as molecular triggers of our nanodevices. The nucleic acid cargo strand (orange) is labelled with a fluorophore/quencher pair to easily follow its load/release from the nanomachine. (b,c) Kinetic profiles show triplex complex formation and subsequent cargo release at different concentrations of anti-Dig antibody. (d) The approach is highly specific and works well also in 90% serum (orange bar). (e) We can achieve reversible load and release of the molecular cargo by cyclically adding anti-Dig antibody and free Dig in a solution containing both the nanomachine and the cargo strand. (f–j) Comparable efficiency and results can be achieved using a nanomachine that is labelled with two molecules of DNP at the two ends and thus triggered with anti-DNP antibodies. (k) The two nanomachines can orthogonally work in the same solution without crosstalk. (l) Moreover, the cargo strand displaced on antibody binding can activate a toehold strand-displacement reaction. The experiments shown in this and in the following figures were performed in 50 mM Na₂HPO₄, 150 mM NaCl and 10 mM MgCl₂ at pH 6.8, 37 °C at an equimolar (50 nM) concentration of nanomachine and cargo unless otherwise noted. Cycles' experiments were performed adding the concentration of antibody indicated in e,j and a concentration of 300 nM of free Dig or DNP. The experimental values represent mean±s.d. of three separate measurements.

Figure 4. Modular antibody-powered DNA nanomachine.

Modular nanomachines employing three different antigens: digoxigenin (a–e), dinitrophenol (DNP) (f–j) and a 12-residue epitope (p17 peptide) excised from the HIV-1 matrix protein (k–o). All these nanomachines are triggered by their specific target antibodies while exhibiting no significant response to high concentrations of the non-specific targets. (p) Such modular antibody-powered nanomachine can be adapted to an AND-logic gate that releases its cargo only in the simultaneous presence of two different antibodies. To demonstrate this, we modified a modular nanomachine with the recognition elements Dig and DNP. (q) Due to the steric hindrance mechanism that disrupts triplex-forming interactions, we observe the cargo release only in the simultaneous presence of both anti-Dig and anti-DNP antibodies. (r) The modular antibody-powered nanomachine also allows to reversibly change the recognition element on the fly via the displacement and substitution of the antigen-conjugated strand (orange and grey). By doing so we can achieve a controlled release of the DNA cargo with two distinct antibodies in the same solution. The experiments reported here were performed in 50 mM Na₂HPO₄, 150 mM NaCl and 10 mM MgCl₂ at pH 6.8, 37 °C at an equimolar (50 nM) concentration of nanomachine, each antigen conjugated strand and cargo. Cycles' experiments were performed adding a concentration of 100 nM of antibody and a concentration of 300 nM of Dig, DNP and p17 peptide. The experimental values represent mean±s.d. of three separate measurements.