High-Affinity Host-Guest Recognition for Efficient Assembly and Enzymatic Responsiveness of DNA Nanostructures.

The combination of multiple orthogonal interactions enables hierarchical complexity in self-assembled nanoscale materials. Here, efficient supramolecular polymerization of DNA origami nanostructures is demonstrated using a multivalent display of small molecule host-guest interactions. Modification of DNA strands with cucurbit[7]uril (CB[7]) and its adamantane guest, yielding a supramolecular complex with an affinity of order 1010 m-1 , directs hierarchical assembly of origami monomers into 1D nanofibers. This affinity regime enables efficient polymerization; a lower-affinity ß-cyclodextrin-adamantane complex does not promote extended structures at a similar valency. Finally, the utility of the high-affinity CB[7]-adamantane interactions is exploited to enable responsive enzymatic actuation of origami nanofibers assembled using peptide linkers. This work demonstrates the power of high-affinity CB[7]-guest recognition as an orthogonal axis to drive self-assembly in DNA nanotechnology.

Organic-inorganic composite of polypropylene fumarate and nanohydroxyapatite as carrier of antibiotics for the treatment of bone infections.

Current treatment approaches in clinics to treat the infectious lesions have partial success thus demanding the need for development of advanced treatment modalities. In this study we fabricated an organic-inorganic composite of polypropylene fumarate (PPF) and nanohydroxyapatite (nHAP) by photo-crosslinking as a carrier of two clinically used antibiotics, ciprofloxacin (CIP) and rifampicin (RFP) for the treatment of bone infections. Carboxy terminal-PPF was first synthesized by cis-trans isomerization of maleic anhydride which was then photo-crosslinked using diethylfumarate (DEF) as crosslinker and bis-acylphosphine oxide (BAPO) as photo-initiator under UV lights (P). A composite of PPF and nHAP was fabricated by incorporating 40 % of nHAP in the polymeric matrix of PPF (PH) which was then characterized for different physicochemical parameters. CIP was added along with nHAP to fabricated CIPloaded composite scaffolds (PHC) which was then coated with RFP to synthesize RFP coated CIP-loaded scaffolds (PHCR). It was observed that there was a temporal separation in the in vitro release of two antibiotics after coating PHC with RFP with 80.48 ± 0.40 % release of CIP from PHC and 62.43 ± 0.21 % release of CIP from PHCR for a period of 60 days. Moreover, in vitro protein adsorption was also found to be maximum in PHCR (154.95 ± 0.07 µg/mL) as observed in PHC (75.42 ± 0.06 µg/mL), PH (24.47 ± 0.08 µg/mL) and P alone (4.47 ± 0.02 µg/mL). The scaffolds were also evaluated using in vivo infection model to assess their capacity in reducing the bacterial burden at the infection site. The outcome of this study suggests that RFP coated CIP-loaded PPF composite scaffolds could reduce bacterial burden and simultaneously augment bone healing during infection related fractures.

CytoDirect: A Nucleic Acid Nanodevice for Specific and Efficient Delivery of Functional Payloads to the Cytoplasm.

Direct and efficient delivery of functional payloads such as chemotherapy drugs, siRNA, or small-molecule inhibitors into the cytoplasm, bypassing the endo/lysosomal trapping, is a challenging task for intracellular medicine. Here, we take advantage of the programmability of DNA nanotechnology to develop a DNA nanodevice called CytoDirect, which incorporates disulfide units and human epidermal growth factor receptor 2 (HER2) affibodies into a DNA origami nanostructure, enabling rapid cytosolic uptake into targeted cancer cells and deep tissue penetration. We further demonstrated that therapeutic oligonucleotides and small-molecule chemotherapy drugs can be easily delivered by CytoDirect and showed notable effects on gene knockdown and cell apoptosis, respectively. This study demonstrates the synergistic effect of disulfide and HER2 affibody modifications on the rapid cytosolic delivery of DNA origami and its payloads to targeted cells and deep tissues, thereby expanding the delivery capabilities of DNA nanostructures in a new direction for disease treatment.

High-affinity binding to the SARS-CoV-2 spike trimer by a nanostructured, trivalent protein-DNA synthetic antibody.

Multivalency enables nanostructures to bind molecular targets with high affinity. Although antibodies can be generated against a wide range of antigens, their shape and size cannot be tuned to match a given target. DNA nanotechnology provides an attractive approach for designing customized multivalent scaffolds due to the addressability and programmability of the nanostructure shape and size. Here, we design a nanoscale synthetic antibody ("nano-synbody") based on a three-helix bundle DNA nanostructure with one, two, or three identical arms terminating in a mini-binder protein that targets the SARS-CoV-2 spike protein. The nano-synbody was designed to match the valence and distance between the three receptor binding domains (RBDs) in the spike trimer, in order to enhance affinity. The protein-DNA nano-synbody shows tight binding to the wild-type, Delta, and several Omicron variants of the SARS-CoV-2 spike trimer, with affinity increasing as the number of arms increases from one to three. The effectiveness of the nano-synbody was also verified using a pseudovirus neutralization assay, with the three-arm nanostructure inhibiting two Omicron variants against which the structures with only one or two arms are ineffective. The structure of the three-arm nano-synbody bound to the Omicron variant spike trimer was solved by negative-stain transmission electron microscopy reconstruction, and shows the protein-DNA nanostructure with all three arms attached to the RBD domains, confirming the intended trivalent attachment. The ability to tune the size and shape of the nano-synbody, as well as its potential ability to attach two or more different binding ligands, will enable the high-affinity targeting of a range of proteins not possible with traditional antibodies.

Automated design of 3D DNA origami with non-rasterized 2D curvature.

Improving the precision and function of encapsulating three-dimensional (3D) DNA nanostructures via curved geometries could have transformative impacts on areas such as molecular transport, drug delivery, and nanofabrication. However, the addition of non-rasterized curvature escalates design complexity without algorithmic regularity, and these challenges have limited the ad hoc development and usage of previously unknown shapes. In this work, we develop and automate the application of a set of previously unknown design principles that now includes a multilayer design for closed and curved DNA nanostructures to resolve past obstacles in shape selection, yield, mechanical rigidity, and accessibility. We design, analyze, and experimentally demonstrate a set of diverse 3D curved nanoarchitectures, showing planar asymmetry and examining partial multilayer designs. Our automated design tool implements a combined algorithmic and numerical approximation strategy for scaffold routing and crossover placement, which may enable wider applications of general DNA nanostructure design for nonregular or oblique shapes.

Coarse-Grained Simulations for the Characterization and Optimization of Hybrid Protein-DNA Nanostructures.

We present here the combination of experimental and computational modeling tools for the design and characterization of protein-DNA hybrid nanostructures. Our work incorporates several features in the design of these nanostructures: (1) modeling of the protein-DNA linker identity and length; (2) optimizing the design of protein-DNA cages to account for mechanical stresses; (3) probing the incorporation efficiency of protein-DNA conjugates into DNA nanostructures. The modeling tools were experimentally validated using structural characterization methods like cryo-TEM and AFM. Our method can be used for fitting low-resolution electron density maps when structural insights cannot be deciphered from experiments, as well as enable in-silico validation of nanostructured systems before their experimental realization. These tools will facilitate the design of complex hybrid protein-DNA nanostructures that seamlessly integrate the two different biomolecules.

Current strategies in tailoring methods for engineered exosomes and future avenues in biomedical applications.

Exosomes are naturally occurring nanovesicles of endosomal origin, responsible for cellular communication. Depending on the cell type, exosomes display disparity in the cargo and are involved in up/down regulation of different biological pathways. Naturally secreted exosomes, owing to their inherent delivery potential, non-immunogenic nature and limited structural resemblance to the cells have emerged as ideal candidates for various drug delivery and therapeutic applications. Moreover, the structural versatility of exosomes provides greater flexibility for surface modifications to be made in the native configuration, by different methods, like genetic-engineering, chemical procedures, physical methods and microfluidic-technology, to enhance the cargo quality for expanded biomedical applications. Post isolation and prior to engineering exosomes for various applications, the internal and external structural compositions of exosomes are studied via different techniques. Efficiency and scalability of the exosome modification methods are pivotal in determining the scope of the technique for clinical applications. This review majorly focuses on different methods employed for engineering exosomes, and advantages/disadvantages associated with different tailoring approaches, along with the efficacy of engineered exosomes in biomedical applications. Further, the review highlights the importance of a relatively recent avenue for delivery of exosomes via scaffold-based delivery of naïve/engineered exosomes for regenerative medicine and tissue engineering. This review is based on the recent knowledge generated in this field and our comprehension in this domain.