Design, construction and characterization of aptamer-functionalized DNA origami for malaria diagnosis

Abstract

DNA origami technique allows for fabrication of nanostructures in a wide variety of shapes. The programmability of DNA origami structure is achieved via self-assembly of single-stranded DNA scaffolds with the assistance of numerous DNA staples. Due to the programmability of unique DNA sequences of individual DNA staples, DNA origamis may be functionalized and chemically modified with nanoscale precision. This intrinsic feature of DNA origamis enables the development of various sophisticated DNA-origami based nanomechanical devices.

DNA aptamers are versatile tools for recognition of specific biomolecules. Due to their utility, aptamers have been integrated extensively into different biosensor platforms for analytical purposes. However, little research has been done into the synergy of these DNA technologies or their potential applications in nanoscale disease therapeutics and diagnostics.

In light of this, the DNA aptamer 2008s, which binds specifically to malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) was integrated into DNA origami structures in order to create nanoscale malaria diagnostic devices. To facilitate this, an aptamer lock duplex was created by hybridizing 2008s with complementary oligonucleotides, both of which were subsequently incorporated into a DNA origami box such that closed origami box was unlocked and opened in response to the aptamer lock strand displacement triggered by PfLDH. To create a lock which is stable yet amenable to being unlocked, the aptamer lock design was optimized by adjusting the number of base pairs inside the aptamer lock duplex as well as the position where complementary DNA would be annealed to 2008s. The resulting aptamer lock named “12bp1” could bind effectively and specifically with PfLDH. In parallel, the folding mechanism of a 36 nm x 36 nm x 42 nm DNA origami box was studied and origami assembly protocols were optimized. Microscopic visualization using transmission electron microscopy confirmed the integrity of the assembled DNA origami box. A pair of 12bp1 aptamer lock duplex were integrated into the DNA origami box at the edge between its face B and D. The aptamer lock functionalized DNA origami box was characterized and its function was demonstrated by incubation with PfLDH, revealing the unlocking and opening of the modified DNA origami boxes. In this study, a functional DNA origami box, combined with a pair of 2008s lock duplex, was successfully constructed and well characterized by electron microscopic techniques. This work lays a solid foundation for future development of diagnostics devices based on hybrids of DNA origami and DNA aptamer and demonstrates the utility as well as practicality by which functional DNA aptamers can be integrated into complex DNA nanostructures.