Programmable polymer materials empowered by DNA nanotechnology

Over the past century, synthetic polymers have had a transformative impact on human life, replacing nature-derived materials in many areas. Yet, despite their many advantages, the structure and function of synthetic polymers still appear rudimentary compared to biological matter: cells use dynamic self-assembly to construct complex materials and operate sophisticated macromolecular devices. The field of DNA nanotechnology has demonstrated that synthetic DNA molecules can be programmed to undergo predictable self-assembly, offering unparalleled control over the formation and dynamic properties of artificial nanostructures. Intriguingly, the principles of DNA nanotechnology can be applied to the engineering of soft programmable materials, bringing the abilities of synthetic polymers closer to their biological counterparts. In this perspective, we discuss the unique features of DNA-functionalized polymer materials. We describe design principles that allow researchers to build complex supramolecular architectures with predictable and dynamically adjustable material properties. Finally, we highlight two key application areas where this biologically inspired material class offers particularly promising opportunities: (1) as dynamic matrices for 3D cell and organoid culture and (2) as smart materials for nucleic acid sequencing and pathogen detection.

DNA-scaffolded multivalent vaccine against SARS-CoV-2.

Short peptides are poor immunogens. One way to increase their immune responses is by arraying immunogens in multivalency. Simple and efficient scaffolds for spatial controlling the inter-antigen distance and enhancing immune activation are required. Here, we report a molecular vaccine design principle that maximally drives potent SARS-CoV-2 RBD subunit vaccine on DNA duplex to induce robust and efficacious immune responses in vivo. We expect that the DNA-peptide epitope platform represents a facile and generalizable strategy to enhance the immune response. STATEMENT OF SIGNIFICANCE: DNA scaffolds offer a biocompatible and convenient platform for arraying immunogens in multivalency antigenic peptides, and spatially control the inter-antigen distance. This can effectively enhance immune response. Peptide (instead of entire protein) vaccines are highly attractive. However, short peptides are poor immunogens. Our DNA scaffolded multivalent peptide immunogen system induced robust and efficacious immune response in vivo as demonstrated by the antigenic peptide against SARS-CoV-2. The present strategy could be readily generalized and adapted to prepare multivalent vaccines against other viruses or disease. Particularly, the different antigens could be integrated into one single vaccine and lead to super-vaccines that can protect the host from multiple different viruses or multiple variants of the same virus.

Spatial- and Valence-Matched Neutralizing DNA Nanostructure Blocks Wild-Type SARS-CoV-2 and Omicron Variant Infection.

Natural ligand-receptor interactions that play pivotal roles in biological events are ideal models for design and assembly of artificial recognition molecules. Herein, aiming at the structural characteristics of the spike trimer and infection mechanism of SARS-CoV-2, we have designed a DNA framework-guided spatial-patterned neutralizing aptamer trimer for SARS-CoV-2 neutralization. The ∼5.8 nm tetrahedral DNA framework affords precise spatial organization and matched valence as four neutralizing aptamers (MATCH-4), which matches with nanometer precision the topmost surface of SARS-CoV-2 spike trimer, enhancing the interaction between MATCH-4 and spike trimer. Moreover, the DNA framework provides a dimensionally complementary nanoscale barrier to prevent the spike trimer-ACE2 interaction and the conformational transition, thereby inhibiting SARS-CoV-2-host cell fusion and infection. As a result, the spatial- and valence-matched MATCH-4 ensures improved binding affinity and neutralizing activity against SARS-CoV-2 and its varied mutant strains, particularly the current Omicron variant, that are evasive of the majority of existing neutralizing antibodies. In addition, because neutralizing aptamers specific to other targets can be evolved and assembled, the present design has the potential to inhibit other wide-range and emerging pathogens.

Label-free optical biosensors in the pandemic era

The research in the field of optical biosensors is continuously expanding, thanks both to the introduction of brand new technologies and the ingenious use of established methods. A new awareness on the potential societal impact of this research has arisen as a consequence of the Covid-19 pandemic. The availability of a new generation of analytical tools enabling a more accurate understanding of bio-molecular processes or the development of distributed diagnostic devices with improved performance is now in greater demand and more clearly envisioned, but not yet achieved. In this review, we focus on emerging innovation opportunities conveyed by label-free optical biosensors. We review the most recent innovations in label-free optical biosensor technology in consideration of their competitive potential in selected application areas. The operational simplicity implicit to label-free detection can be exploited in novel rapid and compact devices for distributed diagnostic applications. The adaptability to any molecular recognition or conformational process facilitates the integration of DNA nanostructures carrying novel functions. The high sensitivity to nanoscale objects stimulates the development of ultrasensitive systems down to digital detection of single molecular binding events enhanced by nanoparticles and direct enumeration of bio-nanoparticles like viruses.

Selective Integrin α5ß1 Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles.

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5ß1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.

A Folding-Based Electrochemical Aptasensor for the Single-Step Detection of the SARS-CoV-2 Spike Protein.

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant (KD) value of 43 ± 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available.

Sensitive fluorescence detection of pathogens based on target nucleic acid sequence-triggered transcription.

Accurate identification of mutant pathogens derived from genetic polymorphisms is highly desired in clinical diagnosis. However, current detection methods based on Watson-Crick hybridization suffers from false positives due to the cross-reactivity of wild-type sequences. In this study, we developed an accurate identification of mutant pathogens by combining programmable DNAzyme and target nucleic acid sequence-triggered transcription. Single nucleotide variants (SNVs) are the most plentiful type of mutations in the genome. High specificity to discriminate SNV was first achieved by rational design of dual-hairpin DNA structure and DNAzyme's capability of site-specific cleavage. T7 RNA polymerase-mediated transcription amplification was introduced to exponentially increase the sensitivity by encompassing T7 promoter sequence into the dual-hairpin DNA structure. The design of this biosensor is fast and straightforward without many computational steps, and the highly sensitive biosensor can detect not only SNVs but also occasional insertions and large deletions in the genome. We showed that the assay could rapidly detect COVID-19 variant and methicillin-resistant Staphylococcus aureus (MRSA), and the limit of detection is 0.96 copy/µL. The modular design of functional DNA enables this biosensor be easily reconfigured and is useful diagnosis of emerging infectious diseases caused by mutant pathogens.

Self-resetting molecular probes for nucleic acids detection enabled by fuel dissipative systems.

A once-in-a-century global public health crisis, the COVID-19 pandemic has damaged human health and world economy greatly. To help combat the virus, we report a self-resetting molecular probe capable of repeatedly detecting SARS-CoV-2 RNA, developed by orchestrating a fuel dissipative system via DNA nanotechnology. A set of simulation toolkits was utilized to design the probe, permitting highly consistent signal amplitudes across cyclic detections. Uniquely, full width at half maximum regulated by dissipative kinetics exhibits a fingerprint signal suitable for high confidential identifications of single-nucleotide variants. Further examination on multiple human-infectious RNA viruses, including ZIKV, MERS-CoV, and SARS-CoV, demonstrates the generic detection capability and superior orthogonality of the probe. It also correctly classified all the clinical samples from 55 COVID-19 patients and 55 controls. Greatly enhancing the screening capability for COVID-19 and other infectious diseases, this probe could help with disease control and build a broader global public health agenda.

Sequential Therapy of Acute Kidney Injury with a DNA Nanodevice.

The high demand for acute kidney injury (AKI) therapy calls the development of multifunctional nanomedicine for renal management with programmable pharmacokinetics. Here, we developed a renal-accumulating DNA nanodevice with exclusive kidney retention for longitudinal protection of AKI in different stages in a renal ischemia-reperfusion (I/R) model. Due to the prolonged kidney retention time (>12 h), the ROS-sensitive nucleic acids of the nanodevice could effectively alleviate oxidative stress by scavenging ROS in stage I, and then the anticomplement component 5a (aC5a) aptamer loaded nanodevice could sequentially suppress the inflammatory responses by blocking C5a in stage II, which is directly related to the cytokine storm. This sequential therapy provides durable and pathogenic treatment of kidney dysfunction based on successive pathophysiological events induced by I/R, which holds great promise for renal management and the suppression of the cytokine storm in more broad settings including COVID-19.

DNA-inspired nanomaterials for enhanced endosomal escape.

To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a "proton sponge" effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.