Aptamer-enabled DNA nanostructures for biomedical applications

Abstract

DNA nanotechnology uses nucleic acids to fabricate tools for biomedical applications. There are several advantages of using DNA nanostructures for diagnostics including novel mechanisms of detection and low costs of production and stability. To allow detection of different biomarkers, it is useful to incorporate aptamers into the design. In my PhD, five different strategies were developed to use DNA nanostructures for biomedical applications. Three of them were incorporated with a malaria specific aptamer allowing them to detect the presence of Plasmodium falciparum lactate dehydrogenase (PfLDH).

The first nanodevice was a design of DNA nanotweezers. The single-stranded PfLDH aptamer was split into two fragments and incorporated into the three-stranded nanotweezers as target recognition sites. Presence of PfLDH closed the nanotweezers and induced the formation of complete G-quadruplex. The quadruplex then bound to hemin for increasing the peroxidase activity that led to an observable and colorimetric signal. Because of the size difference between nucleic acid and protein, the spatial flexibility of nanotweezers was optimized for the maximum structural changing efficiency.

An opening mechanism was also developed by incorporating the aptamer to a DNA origami nanobox. It was done by first optimizing the complementarity between aptamer and a complementary strand for the highest strand-displacement efficiency by PfLDH. The duplex was used as a lock to be integrated to the opening of nanobox. The presence of PfLDH displaced the aptamer and successfully opened the box. It could be observed by electron microscopy and reduction in fluorescence resonance energy transfer (FRET) from a FRET pair also attached to the opening. The opening of nanobox also indicated the potential of using this design for targeted drug delivery.

For clinical diagnosis, the previous two designs distorted the original structure of aptamer that affected the affinity towards PfLDH. So, DNA polyhedra were designed to support aptamer for enhancing sensitivity. The use of polyhedra elevated the aptamer from the coated surface. The elevation allowed the aptamer to be more accessible to PfLDH in solution sample. Tetrahedron was found to be the most effective among all designs. A DNA tile was also designed for signal amplification. The presence of target would induce the formation two-dimensional tile increasing the molecular ratio between target and signal reporter as amplification for improving the design of DNA nanotweezers.

A novel DNA nanostructure called mazzocchio was also designed as a potential carrier for drug delivery. It possessed the advantage of biocompatibility of DNA origami while solving the issue of complexity in synthesis. It consisted of repeating units made from 12 single-stranded DNAs. The evidences on gel electrophoresis and electron microscopy proved that it was successful in inducing curvature during the polymerization of subunits and the mazzocchio was made with the expected dimension.

The work in my PhD demonstrated the potential of using DNA nanostructure for clinical applications especially with the functionalization of DNA aptamer. Further development will lead to the possibility of replacing current strategies which typically use antibodies. Such approaches may transform diagnostics and therapeutics to benefit society.