Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures.

The regulation of thrombin activity offers an opportunity to regulate blood clotting because of the central role played by this molecule in the coagulation cascade. Thrombin-binding DNA aptamers have been used to inhibit thrombin activity. In the past, to address the low efficacy reported for these aptamers during clinical trials, multiple aptamers have been linked using DNA nanostructures. Here, we modify that strategy by linking multiple copies of various thrombin-binding aptamers using DNA weave tiles. The resulting constructs have very high anticoagulant activity in functional assays owing to their improved cooperative binding affinity to thrombin due to optimized spacing, orientation, and the high local concentration of aptamers. We also report the results of molecular dynamics simulations to gain insight into the solution conformations of the tiles. Moreover, by using DNA strand displacement, we were able to turn the coagulation cascade off and on as desired, thereby enabling significantly better control over blood coagulation.

Building DNA nanostructures for molecular computation, templated assembly, and biological applications.

CONSPECTUS: DNA is a critical biomolecule well-known for its roles in biology and genetics. Moreover, its double-helical structure and the Watson-Crick pairing of its bases make DNA structurally predictable. This predictability enables design and synthesis of artificial DNA nanostructures by suitable programming of the base sequences of DNA strands. Since the advent of the field of DNA nanotechnology in 1982, a variety of DNA nanostructures have been designed and used for numerous applications. In this Account, we discuss the progress made by our lab which has contributed toward the overall advancement of the field. Tile-based DNA nanostructures are an integral part of structural DNA nanotechnology. These structures are formed using several short, chemically synthesized DNA strands by programming their base sequences so that they self-assemble into desired constructs. Design and assembly of several DNA tiles will be discussed in this Account. Tiles include, for example, TX tiles with three parallel, coplanar duplexes, 4 × 4 cross-tiles with four arms, and weave-tiles with weave-like architecture. Another category of tiles we will present involve multiple parallel duplexes that assemble to form closed tubular structures. All of these tile types have been used to form micrometer-scale one- and two-dimensional arrays and lattices. Origami-based structures constitute another category where a long single-stranded DNA scaffold is folded into desired shapes by association with multiple short staple strands. This Account will describe the efforts by our lab in devising new strategies to improve the maximum size of origami structures. The various DNA nanostructures detailed here have been used in a wide variety of different applications. This Account will discuss the use of DNA tiles for logical computation, encoding information as molecular barcodes, and functionalization for patterning of other nanoscale organic and inorganic materials. Consequently, we have used DNA nanostructures for templating metallic nanowires as well as for programmed assembly of proteins and nanoparticles with controlled spacings. Among other applications, we have used DNA nanotechnology in biosensors that detect target DNA sequences and to affect cell surface receptor clustering for communicating with a cell signaling pathway. We used DNA weave-tiles to control the spacing between thrombin-binding aptamers which resulted in very high antithrombin and anticoagulant activity of the construct. We believe that the tremendous progress in DNA nanotechnology over the past three decades will open even more research avenues in the near future for applications in a wide variety of disciplines including electronics, photonics, biomedical engineering, biosensing, therapeutics, and nucleic-acid-based drug delivery.

An autonomously self-assembling dendritic DNA nanostructure for target DNA detection.

There is a growing need for sensitive and reliable nucleic acid detection methods that are convenient and inexpensive. Responsive and programmable DNA nanostructures have shown great promise as chemical detection systems. Here, we describe a DNA detection system employing the triggered self-assembly of a novel DNA dendritic nanostructure. The detection protocol is executed autonomously without external intervention. Detection begins when a specific, single-stranded target DNA strand (T) triggers a hybridization chain reaction (HCR) between two, distinct DNA hairpins (α and ß). Each hairpin opens and hybridizes up to two copies of the other. In the absence of T, α and ß are stable and remain in their poised, closed-hairpin form. In the presence of T, α hairpins are opened by toe-hold mediated strand-displacement, each of which then opens and hybridizes two ß hairpins. Likewise, each opened ß hairpin can open and hybridize two α hairpins. Hence, each layer of the growing dendritic nanostructure can in principle accommodate an exponentially increasing number of cognate molecules, generating a high molecular weight nanostructure. This HCR system has minimal sequence constraints, allowing reconfiguration for the detection of arbitrary target sequences. Here, we demonstrate detection of unique sequence identifiers of HIV and Chlamydia pathogens.

Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures.

Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation. FROM THE CLINICAL EDITOR: Thrombin inhibition via DNA aptamers has recently become a possibility. In this study, thrombin-binding DNA aptamers were further optimized by nanoscale organization on DNA nanostructures. The authors have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than that of free aptamer. Reversal of thrombin inhibition was also achieved by single-stranded DNA antidotes, enabling significant control over the coagulation pathway.

Design and synthesis of DNA four-helix bundles.

The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

Design and construction of double-decker tile as a route to three-dimensional periodic assembly of DNA.

DNA is a useful material for nanoscale construction. Due to highly specific Watson-Crick base pairing, the DNA sequences can be designed to form small tiles or origami. Adjacent helices in such nanostructures are connected via Holliday junction-like crossovers. DNA tiles can have sticky ends which can then be programmed to form large one-dimensional and two-dimensional periodic lattices. Recently, a three-dimensional DNA lattice has also been constructed. Here we report the design and construction of a novel DNA cross tile, called the double-decker tile. Its arms are symmetric and have four double helices each. Using its sticky ends, large two-dimensional square lattices have been constructed which are on the order of tens of micrometers. Furthermore, it is proposed that the sticky ends of the double-decker tile can be programmed to form a three-dimensional periodic lattice with large cavities that could be used as a scaffold for precise positioning of molecules in space.

Weave tile architecture construction strategy for DNA nanotechnology.

Architectural designs for DNA nanostructures typically fall within one of two broad categories: tile-based designs (assembled from chemically synthesized oligonucleotides) and origami designs (woven structures employing a biological scaffold strand and synthetic staple strands). Both previous designs typically contain many Holliday-type multi-arm junctions. Here we describe the design, implementation, and testing of a unique architectural strategy incorporating some aspects of each of the two previous design categories but without multi-arm junction motifs. Goals for the new design were to use only chemically synthesized DNA, to minimize the number of component strands, and to mimic the back-and-forth, woven strand routing of the origami architectures. The resulting architectural strategy employs "weave tiles" formed from only two oligonucleotides as basic building blocks, thus decreasing the burden of matching multiple strand stoichiometries compared to previous tile-based architectures and resulting in a structurally flexible tile. As an example application, we have shown that the four-helix weave tile can be used to increase the anticoagulant activity of thrombin-binding aptamers in vitro.

Retention of enzymatic activity of alpha-amylase in the reductive synthesis of gold nanoparticles.

In this paper, we report the generation of Au nanoparticles (NPs), using a pure enzyme for the reduction of AuCl4(-), with the retention of enzymatic activity in the complex. As a model system, alpha-amylase was used to readily synthesize and stabilize Au NPs in aqueous solution. Although several other enzymes were also pursued for the synthesis, it was interesting to observe that only alpha-amylase and EcoRI could produce Au NPs. Following NP synthesis, the activity of the enzyme was retained in the Au NP-alpha-amylase complex. The presence of Au NPs and alpha-amylase in the complex was established by UV-visible and FT-IR spectroscopy, X-ray diffraction (XRD) and transmission electron microscopic (TEM) measurements. Our observations suggest that the presence of free and exposed S-H groups is essential in the reduction of AuCl4(-) to Au NPs. Structural analysis of the enzymes showed that both alpha-amylase and EcoRI enzymes have free and exposed S-H groups in their native form and thus are suitable for the generation of NPs, whereas the other ones used here do not have such groups. Fortuitously, the enzymatic functional group of alpha-amylase is positioned opposite to that of the free and exposed S-H group, which makes it ideal for the production of Au NPs; binding of the enzyme to Au NPs via Au-S bond and also retention of the biological activity of the enzyme.