Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

DNA-Based MXFs to Enhance Radiotherapy and Stimulate Robust Antitumor Immune Responses.

Metal "X" Frameworks (MXFs) constructed from metal ions and biomacromolecules ("X components") via coordination interactions show crystalline structures and diverse functionalities. Here, a series of MXFs composed of various metal ions (e.g., Zn2+, Hf4+, Ca2+) and DNA oligodeoxynucleotides were reported. With MXF consisting of Hf4+ and CpG oligodeoxynucleotides as the example, we show that such Hf-CpG MXF can achieve high-Z elements-enhanced photon radiotherapy and further trigger robust tumor-specific immune responses, thus showing efficient tumor suppression ability. In vivo experiments showed that external beam radiotherapy applied on tumors locally injected with Hf-CpG MXF result in the thorough elimination of primary tumors, complete inhibition of tumor metastasis, and protection against tumor rechallenge by triggering robust antitumor immune responses. Our findings provide a blueprint for fabricating a variety of rationally designed MXFs with desired functions and present the strategy of stimulating whole-body systemic immune responses by only local treatment of radiotherapy.

Aptamer-Based Logic Computing Reaction on Living Cells to Enable Non-Antibody Immune Checkpoint Blockade Therapy.

Precise and lasting immune checkpoint blockade (ICB) therapy with high objective response rate remains a significant challenge in clinical trials. We thus report the development of an aptamer-based logic computing reaction to covalently conjugate immune checkpoint antagonizing aptamers (e.g., aPDL1 aptamer) on the surface of cancer cells, achieving effective and sustained ICB therapy without the need for antibodies. Specifically, azides were metabolically labeled on the cell-surface glycoproteins as "chemical receptors", enabling cyclooctyne-coupling aPDL1 aptamers to achieve aptamer-based logic computing-mediated azides/cyclooctynes-based bioorthogonal reaction. In stepwise fashion, PDL1 plus azide-bearing glycoproteins are expressed on cells and become multiple inputs in accordance with Boolean logic. Then, if the "AND" conditions of the algorithm are met, cyclooctyne-coupling aptamers are conjugated on the living cell surface, significantly prolonging overall mouse survival by triggering a precise and sustained T cell-mediated antitumor immunotherapy, otherwise not. Our findings indicate that DNA logic computing-mediated cyclooctyne/azide-based bioorthogonal reaction can improve the precision and robustness of ICB therapy, thereby potentially improving the objective response rate.

Enhancing the Nucleolytic Resistance and Bioactivity of Functional Nucleic Acids by Diverse Nanostructures through in Situ Polymerization-Induced Self-assembly.

Functional nucleic acids (FNAs) are garnering tremendous interest owing to their high modularity and unique bioactivity. Three-dimensional FNAs have been developed to overcome the issues of nuclease degradation and limited cell uptake. We have developed a new facile approach to the synthesis of multiple three-dimensional FNA nanostructures by harnessing photo-polymerization-induced self-assembly. Sgc8 aptamer and CpG oligonucleotide were modified as macro chain-transfer reagents to mediate in situ polymerization and self-assembly. Diverse structures, including micelles, rods, and short worms, afford these two FNAs afford these two FNAs with higher nuclease resistance in serum serum, greater cellular uptake efficiency, and increased bioactivity.

Enhanced in Vivo Blood-Brain Barrier Penetration by Circular Tau-Transferrin Receptor Bifunctional Aptamer for Tauopathy Therapy.

The lack of blood-brain barrier (BBB) penetrating ability has hindered the delivery of many therapeutic agents for tauopathy treatment. In this study, we report the synthesis of a circular bifunctional aptamer to enhance the in vivo BBB penetration for better tauopathy therapy. The circular aptamer consists of one reported transferrin receptor (TfR) aptamer to facilitate TfR-aptamer recognition-induced transcytosis across BBB endothelial cells, and one Tau protein aptamer that we recently selected to inhibit Tau phosphorylation and other tauopathy-related pathological events in the brain. This novel circular Tau-TfR bifunctional aptamer displays significantly improved plasma stability and brain exposure, as well as the ability to disrupt tauopathy and improve traumatic brain injury (TBI)-induced cognitive/memory deficits in vivo, providing important proof-of-principle evidence that circular Tau-TfR aptamer can be further developed into diagnostic and therapeutic candidates for tauopathies.

Artificial Signal Feedback Network Mimicking Cellular Adaptivity.

Inspired by this elegant system of cellular adaptivity, we herein report the rational design of a dynamic artificial adaptive system able to sense and respond to environmental stresses in a unique sense-and-respond mode. Utilizing DNA nanotechnology, we constructed an artificial signal feedback network and anchored it to the surface membrane of a model giant membrane vesicle (GMV) protocell. Such a system would need to both senses incoming stimuli and emit a feedback response to eliminate the stimuli. To accomplish this mechanistically, our DNA-based artificial signal system, hereinafter termed DASsys, was equipped with a DNA trigger-induced DNA polymer formation and dissociation machinery. Thus, through a sequential cascade of stimulus-induced DNA strand displacement, DASsys could effectively sense and respond to incoming stimuli. Then, by eliminating the stimulus, the membrane surface would return to its initial state, realizing the formation of a cyclical feedback mechanism. Overall, our strategy opens up a route to the construction of artificial signaling system capable of maintaining homeostasis in the cellular micromilieu, and addresses important emerging challenges in bioinspired engineering.

Aptamer Displacement Reaction from Live-Cell Surfaces and Its Applications.

The DNA strand displacement reaction has had sustained scientific interest in building complicated nucleic acid-based networks. However, extending the fundamental mechanism to more diverse biomolecules in a complex environment remains challenging. Aptamers bind with targeted biomolecules with high affinity and selectivity, thus offering a promising route to link the powers of nucleic acid with diverse cues. Here, we describe three methods that allow facile and efficient displacement reaction of aptamers from the living cell surface using complement DNA (cDNA), toehold-labeled cDNA (tcDNA), and single-stranded binding protein (SSB). The kinetics of the DNA strand displacement reaction is severely affected by complex physicochemical properties of the natural membrane. Toehold-mediated and SSB-mediated aptamer displacement exhibited significantly enhanced kinetics, and they completely removed the aptamer quickly to avoid a false signal caused by aptamer internalization. Because of its simplicity, aptamer displacement enabled detection of membrane protein post-translation and improved selection efficiency of cell-SELEX.

Supramolecularly Engineered Circular Bivalent Aptamer for Enhanced Functional Protein Delivery.

Circular bivalent aptamers (cb-apt) comprise an emerging class of chemically engineered aptamers with substantially improved stability and molecular recognition ability. Its therapeutic application, however, is challenged by the lack of functional modules to control the interactions of cb-apt with therapeutics. We present the design of a ß-cyclodextrin-modified cb-apt (cb-apt-ßCD) and its supramolecular interaction with molecular therapeutics via host-guest chemistry for targeted intracellular delivery. The supramolecular ensemble exhibits high serum stability and enhanced intracellular delivery efficiency compared to a monomeric aptamer. The cb-apt-ßCD ensemble delivers green fluorescent protein into targeted cells with efficiency as high as 80%, or cytotoxic saporin to efficiently inhibit tumor cell growth. The strategy of conjugating ßCD to cb-apt, and subsequently modulating the supramolecular chemistry of cb-apt-ßCD, provides a general platform to expand and diversify the function of aptamers, enabling new biological and therapeutic applications.

Modulating Aptamer Specificity with pH-Responsive DNA Bonds.

Aptamers that recognize specific cells in a complex environment have emerged as invaluable molecular tools in bioanalysis and in the development of targeted therapeutics. The selective recognition of aptamers, however, can be compromised by the coexistence of target receptors on both target cells and other cells. To address this problem, we constructed a structure-switchable aptamer (SW-Apt) with reconfigurable binding affinity in accordance with the microenvironment of target cells. The SW-Apt makes use of i-motifs, which are quadruplex structures that form in sequences rich in cytosine. More specifically, we report the design of single-stranded, pH-responsive i-motif-modified aptamers able to bind specifically with target cells by exploiting their pH. Here, the i-motif serves as a structural domain to either facilitate the binding ability of aptamers to target cells or suppress the binding ability of aptamers to nontarget cell based on the pH of the cellular microenvironment. SW-Apt exhibited high binding ability with target cells at acidic pH, while no obvious binding was observed at physiological pH. The i-motif-induced structure-switching was verified with Förster resonance energy transfer and circular dichroism spectroscopy. Notably, SW-Apt exhibits high specificity in serum and excellent stability, likely attributed to the folded quadruplex i-motif structure. This study provides a simple and efficient strategy to chemically modulate aptamer binding ability and thus improve aptamer binding specificity to target cells, irrespective of the coexistence of identical receptors on target and nontarget cells.

Identification and Characterization of DNA Aptamers Specific for Phosphorylation Epitopes of Tau Protein.

Tau proteins are proteins that stabilize microtubules, but their hyperphosphorylation can result in the formation of protein aggregates and, over time, neurodegeneration. This phenomenon, termed tauopathy, is pathologically involved in several neurodegenerative disorders. DNA aptamers are single-stranded oligonucleotides capable of specific binding to target molecules. Using tau epitopes predisposed for phosphorylation, we identified six distinct aptamers that bind to tau at two phosphorylatable epitopes (Thr-231 and Ser-202) and to full-length Tau441 proteins with nanomolar affinity. In addition, several of these aptamers also inhibit tau phosphorylation (IT4, IT5, IT6) and tau oligomerization (IT3, IT4, IT5, IT6). This is the first report to identify tau epitope-specific aptamers. Such tau aptamers can be used to detect tau in biofluids and uncover the mechanism of tauopathy. They can be further developed into novel therapeutic agents in mitigating tauopathy-associated neurodegenerative disorders.

Self-Assembled Aptamer-Grafted Hyperbranched Polymer Nanocarrier for Targeted and Photoresponsive Drug Delivery.

Photoresponsive materials are emerging as ideal carriers for precisely controlled drug delivery owing to their high spatiotemporal selectivity. However, drawbacks such as slow release kinetics, inherent toxicity, and lack of targeting ability hinder their translation into clinical use. We constructed a new DNA aptamer-grafted photoresponsive hyperbranched polymer, which can self-assemble into nanoparticles, thereby achieving biocompatibility and target specificity, as well as light-controllable release behavior. Upon UV-irradiation, rapid release induced by disassembly was observed for Nile Red-loaded nanoparticles. Further in vitro cell studies confirmed this delivery system's specific binding and internalization performance arising from the DNA aptamer corona. The DOX-loaded nanoassembly exhibited selective phototriggered cytotoxicity towards cancer cells, indicating its promising therapeutic effect as a smart drug delivery system.

Cross-Linked Aptamer-Lipid Micelles for Excellent Stability and Specificity in Target-Cell Recognition.

The specific binding ability of DNA-lipid micelles (DLMs) can be increased by the introduction of an aptamer. However, supramolecular micellar structures based on self-assemblies of amphiphilic DLMs are expected to demonstrate low stability when interacting with cell membranes under certain conditions, which could lead to a reduction in selectivity for targeting cancer cells. We herein report a straightforward cross-linking strategy that relies on a methacrylamide branch to link aptamer and lipid segments. By an efficient photoinduced polymerization process, covalently linked aptamer-lipid units help stabilize the micelle structure and enhance aptamer probe stability, further improving the targeting ability of the resulting nanoassembly. Besides the development of a facile cross-linking method, this study clarifies the relationship between aptamer-lipid concentration and the corresponding binding ability.

Recognition-then-Reaction Enables Site-Selective Bioconjugation to Proteins on Live-Cell Surfaces.

Site-selective protein modification is a key step in facilitating protein functionalization and manipulation. To accomplish this, genetically engineered proteins were previously required, but the procedure was laborious, complex, and technically challenging. Herein we report the development of aptamer-based recognition-then-reaction to guide site-selective protein/DNA conjugation in a single step with outstanding selectivity and efficiency. As models, several proteins, including human thrombin, PDGF-BB, Avidin, and His-tagged recombinant protein, were studied, and the results showed excellent selectivity under mild reaction conditions. Taking advantage of aptamers as recognition elements with extraordinary selectivity and affinity, this simple preparation method can tag a protein in a complex milieu. Thus, with the aptamer obtained from cell-SELEX, real-time modification of live-cell membrane proteins can be achieved in one step without any pre-treatment.

Method to study stoichiometry of protein post-translational modification.

We report a new method to study stoichiometry of protein post-translational modification (PTM) via manipulating small-molecule labels. Such labels can simultaneously bind with all the modified amino acids in a target protein, resulting in a large signal. However, if the small-molecule labels are previously attached with a macromolecule comparable to the target protein in size, binding of one such label will prevent subsequent binding of more labels of its kind; thus, only one of the modified amino acids in the target protein can be bound with the label, resulting in a small signal. Therefore, the stoichiometric number of PTM can be determined by a ratio of the two signals. The proposed method can analyze nitration of several essential regulatory proteins of cellular life, all of which are found to have an integer stoichiometric number indicating regulated site-specific nitration. These results validate the efficacy of our method to provide detailed information on PTMs with biological significance.

Extracellular vesicles for developing targeted hearing loss therapy.

Substantial efforts have been made for local administration of small molecules or biologics in treating hearing loss diseases caused by either trauma, genetic mutations, or drug ototoxicity. Recently, extracellular vesicles (EVs) naturally secreted from cells have drawn increasing attention on attenuating hearing impairment from both preclinical studies and clinical studies. Highly emerging field utilizing diverse bioengineering technologies for developing EVs as the bioderived therapeutic materials, along with artificial intelligence (AI)-based targeting toolkits, shed the light on the unique properties of EVs specific to inner ear delivery. This review will illuminate such exciting research field from fundamentals of hearing protective functions of EVs to biotechnology advancement and potential clinical translation of functionalized EVs. Specifically, the advancements in assessing targeting ligands using AI algorithms are systematically discussed. The overall translational potential of EVs is reviewed in the context of auditory sensing system for developing next generation gene therapy.

Proteolysis targeting chimera extracellular vesicles for therapeutic development treating triple negative breast cancer.

Proteolysis targeting chimeras (PROTACs) are an emerging targeted cancer therapy approach, but wide-spread clinical use of PROTAC is limited due to poor cell targeting and penetration, and instability in vivo. To overcome such issues and enhance the in vivo efficacy of PROTAC drugs, microfluidic droplet-based electroporation (µDES) was developed as a novel extracellular vesicle (EVs) transfection system, which enables the high-efficient PROTAC loading and effective delivery in vivo. Our previously developed YX968 PROTAC drug had shown the selectively degradation of HDAC3 and 8, which effectively suppresses the growth of breast tumor cell lines, including MDA-MB-231 triple negative breast cancer (TNBC) line, via dual degradation without provoking a global histone hyperacetylation. In this study, we demonstrated that µDES-based PROTAC loading in EVs significantly enhanced therapeutic function of PROTAC drug in vivo in the TNBC breast tumor mouse model. NSG mice with pre-established MDA-MB-231 tumors and treated with intraperitoneal injection of EVs for tumor inhibition study, which showed significantly higher HDAC 3 and 8 degradation efficiency and tumor inhibition than PROTAC only group. The liver, spleen, kidney, lung, heart, and brain were collected for safety testing, which exhibited improved toxicity. The EV delivery of PROTAC drug enhances drug stability and bioavailability in vivo, transportability, and drug targeting ability, which fills an important gap in current development of PROTAC therapeutic functionality in vivo and clinical translation. This novel EV-based drug transfection and delivery strategy could be applicable to various therapeutics for enhancing in vivo delivery, efficacy, and safety.

A folding motif formed with an expanded genetic alphabet.

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ-:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ-:dZ pairs join parallel strands in a four-stranded compact down-up-down-up fold. These have two possible structures: one with intercalated dZ-:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis.

CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss.

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (µDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR).

Extracellular Vesicles as an Advanced Delivery Biomaterial for Precision Cancer Immunotherapy.

In recent years, cancer immunotherapy has been observed in numerous preclinical and clinical studies for showing benefits. However, due to the unpredictable outcomes and low response rates, novel targeting delivery approaches and modulators are needed for being effective to more broader patient populations and cancer types. Compared to synthetic biomaterials, extracellular vesicles (EVs) specifically open a new avenue for improving the efficacy of cancer immunotherapy by offering targeted and site-specific immunity modulation. In this review, the molecular understanding of EV cargos and surface receptors, which underpin cell targeting specificity and precisely modulating immunogenicity, are discussed. Unique properties of EVs are reviewed in terms of their surface markers, intravesicular contents, intrinsic immunity modulatory functions, and pharmacodynamic behavior in vivo with tumor tissue models, highlighting key indications of improved precision cancer immunotherapy. Novel molecular engineered strategies for reprogramming and directing cancer immunotherapeutics, and their unique challenges are also discussed to illuminate EV's future potential as a cancer immunotherapeutic biomaterial.

Engineering G-quadruplex aptamer to modulate its binding specificity.

The use of aptamers in bioanalytical and biomedical applications exploits their ability to recognize cell surface protein receptors. Targeted therapeutics and theranostics come to mind in this regard. However, protein receptors occur on both cancer and normal cells; as such, aptamers are now taxed with identifying high vs. low levels of protein expression. Inspired by the flexible template mechanism and elegant control of natural nucleic acid-based structures, we report an allosteric regulation strategy for constructing a structure-switching aptamer for enhanced target cell recognition by engineering aptamers with DNA intercalated motifs (i-motifs) responsive to the microenvironment, such as pH. Structure-switching sensitivity can be readily tuned by manipulating i-motif sequences. However, structure-switching sensitivity is difficult to estimate, making it equally difficult to effectively screen modified aptamers with the desired sensitivity. To address this problem, we selected a fluorescent probe capable of detecting G-quadruplex in complicated biological media.