An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa.

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization.

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

Development of an aptamer capable of multidrug resistance reversal for tumor combination chemotherapy.

Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.

AptaDB: a comprehensive database integrating aptamer-target interactions.

Aptamers have emerged as research hotspots of the next generation due to excellent performance benefits and application potentials in pharmacology, medicine, and analytical chemistry. Despite the numerous aptamer investigations, the lack of comprehensive data integration has hindered the development of computational methods for aptamers and the reuse of aptamers. A public access database named AptaDB, derived from experimentally validated data manually collected from the literature, was hence developed, integrating comprehensive aptamer-related data, which include six key components: (i) experimentally validated aptamer-target interaction information, (ii) aptamer property information, (iii) structure information of aptamer, (iv) target information, (v) experimental activity information, and (vi) algorithmically calculated similar aptamers. AptaDB currently contains 1350 experimentally validated aptamer-target interactions, 1230 binding affinity constants, 1293 aptamer sequences, and more. Compared to other aptamer databases, it contains twice the number of entries found in available databases. The collection and integration of the above information categories is unique among available aptamer databases and provides a user-friendly interface. AptaDB will also be continuously updated as aptamer research evolves. We expect that AptaDB will become a powerful source for aptamer rational design and a valuable tool for aptamer screening in the future. For access to AptaDB, please visit http://lmmd.ecust.edu.cn/aptadb/.

Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

A Poly Aptamer Encoded DNA Nanocatcher Informs Efficient Virus Trapping.

Broad-spectrum antiviral platforms are always desired but still lack the ability to cope with the threats to global public health. Herein, we develop a poly aptamer encoded DNA nanocatcher platform that can trap entire virus particles to inhibit infection with a broad antiviral spectrum. Ultralong single-stranded DNA (ssDNA) containing repeated aptamers was synthesized as the scaffold of a nanocatcher via a biocatalytic process, wherein mineralization of magnesium pyrophosphate on the ssDNA could occur and consequently lead to the formation of nanocatcher with interfacial nanocaves decorated with virus-binding aptamers. Once the viruses were recognized by the apatmers, they would be captured and trapped in the nanocaves via multisite synergistic interactions. Meanwhile, the size of nanocatchers was optimized to prevent their cellular uptake, which further guaranteed inhibition of virus infection. By taking SARS-CoV-2 variants as a model target, we demonstrated the broad virus-trapping capability of a DNA nanocatcher in engulfing the variants and blocking the infection to host cells.

Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

Accurate Cancer Screening and Prediction of PD-L1-Guided Immunotherapy Efficacy Using Quantum Dot Nanosphere Self-Assembly and Machine Learning.

Accurate quantification of exosomal PD-L1 protein in tumors is closely linked to the response to immunotherapy, but robust methods to achieve high-precision quantitative detection of PD-L1 expression on the surface of circulating exosomes are still lacking. In this work, we developed a signal amplification approach based on aptamer recognition and DNA scaffold hybridization-triggered assembly of quantum dot nanospheres, which enables bicolor phenotyping of exosomes to accurately screen for cancers and predict PD-L1-guided immunotherapeutic effects through machine learning. Through DNA-mediated assembly, we utilized two aptamers for simultaneous ultrasensitive detection of exosomal antigens, which have synergistic roles in tumor diagnosis and treatment prediction, and thus, we achieved better sample classification and prediction through machine-learning algorithms. With a drop of blood, we can distinguish between different cancer patients and healthy individuals and predict the outcome of immunotherapy. This approach provides valuable insights into the development of personalized diagnostics and precision medicine.

Toward the Ultimate Limit of Analyte Detection, in Graphene-Based Field-Effect Transistors.

The ultimate sensitivity of field-effect-transistor (FET)-based devices for ionic species detection is of great interest, given that such devices are capable of monitoring single-electron-level modulations. It is shown here, from both theoretical and experimental perspectives, that for such ultimate limits to be approached the thermodynamic as well as kinetic characteristics of the (FET surface)-(linker)-(ion-receptor) ensemble must be considered. The sensitivity was probed in terms of optimal packing of the ensemble, through a minimal charge state/capacitance point of view and atomic force microscopy. Through the fine-tuning of the linker and receptor interaction with the sensing surface, a record limit of detection as well as specificity in the femtomolar range, orders of magnitude better than previously obtained and in excellent accord with prediction, was observed.

Crossing the Halfway Point: Aptamer-Based, Highly Multiplexed Assay for the Assessment of the Proteome.

Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.

Preclinical evaluation of an 18F-labeled Tenascin-C aptamer for PET imaging of atherosclerotic plaque in mouse models of atherosclerosis.

Tenascin-C is an extracellular matrix glycoprotein strongly expressed in coronary atherosclerotic plaque. Aptamers are single-stranded oligonucleotides that bind to specific target molecules with high affinity. This study hypothesized that tenascin-C expression at atherosclerotic plaque in vivo could be detected by tenascin-C specific aptamers using positron emission tomography (PET). This paper reports the radiosynthesis of a fluorine-18 (18F)-labeled tenascin-C aptamer for the biodistribution and PET imaging of the tenascin-C expression in apolipoprotein E-deficient (ApoE-/-) mice. The aortas ApoE-/- mice showed significantly increased positive areas of Oil red O staining than control C57BL/6 mice, and tenascin-C expression was detected in foam cells accumulated in the subendothelial lesions of ApoE-/- mice. The ex vivo biodistribution of the 18F-labeled tenascin-C aptamer showed significantly increased uptake at the aorta of ApoE-/- mice, and ex vivo autoradiography of aorta revealed the high accumulation of the 18F-labeled tenascin-C aptamer in the atherosclerotic lesions of ApoE-/- mice, which was consistent with the location of the atherosclerotic plaques detected by Oil red O staining. PET imaging of the 18F-labeled tenascin-C aptamer revealed a significantly higher mean standardized uptake in the aorta of the ApoE-/- mice than the control C57BL/6 mice. These data highlight the potential use of tenascin-C aptamer to diagnose atherosclerotic lesions in vivo.

Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer.

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.

Proteomics for heart failure risk stratification: a systematic review.

BACKGROUND: Heart failure (HF) is a complex clinical syndrome with persistently high mortality. High-throughput proteomic technologies offer new opportunities to improve HF risk stratification, but their contribution remains to be clearly defined. We aimed to systematically review prognostic studies using high-throughput proteomics to identify protein signatures associated with HF mortality. METHODS: We searched four databases and two clinical trial registries for articles published from 2012 to 2023. HF proteomics studies measuring high numbers of proteins using aptamer or antibody-based affinity platforms on human plasma or serum with outcomes of all-cause or cardiovascular death were included. Two reviewers independently screened articles, extracted data, and assessed the risk of bias. A third reviewer resolved conflicts. We assessed the risk of bias using the Risk Of Bias In Non-randomized Studies-of Exposure tool. RESULTS: Out of 5131 unique articles identified, nine articles were included in the review. The nine studies were observational; three used the aptamer platform, and six used the antibody platform. We found considerable heterogeneity across studies in measurement panels, HF definitions, ejection fraction categorization, follow-up duration, and outcome definitions, and a lack of risk estimates for most protein associations. Hence, we proceeded with a systematic review rather than a meta-analysis. In two comparable aptamer studies in patients with HF with reduced ejection fraction, 21 proteins were identified in common for the association with all-cause death. Among these, one protein, WAP four-disulfide core domain protein 2 was also reported in an antibody study on HFrEF and for the association with CV death. We proposed standardized reporting criteria to facilitate the interpretation of future studies. CONCLUSIONS: In this systematic review of nine studies evaluating the association of proteomics with mortality in HF, we identified a limited number of proteins common across several studies. Heterogeneity across studies compromised drawing broad inferences, underscoring the importance of standardized approaches to reporting.

Role of Exosomes in Cancer and Aptamer-Modified Exosomes as a Promising Platform for Cancer Targeted Therapy.

Exosomes are increasingly recognized as important mediators of intercellular communication in cancer biology. Exosomes can be derived from cancer cells as well as cellular components in tumor microenvironment. After secretion, the exosomes carrying a wide range of bioactive cargos can be ingested by local or distant recipient cells. The released cargos act through a variety of mechanisms to elicit multiple biological effects and impact most if not all hallmarks of cancer. Moreover, owing to their excellent biocompatibility and capability of being easily engineered or modified, exosomes are currently exploited as a promising platform for cancer targeted therapy. In this review, we first summarize the current knowledge of roles of exosomes in risk and etiology, initiation and progression of cancer, as well as their underlying molecular mechanisms. The aptamer-modified exosome as a promising platform for cancer targeted therapy is then briefly introduced. We also discuss the future directions for emerging roles of exosome in tumor biology and perspective of aptamer-modified exosomes in cancer therapy.

A Tumor-Specific Cascade-Activating Smart Prodrug System for Enhanced Targeted Therapy.

Developing intelligently targeted drugs with low side effects is urgent for cancer treatment. Toward this goal, a tumor-specific cascade-activating smart prodrug system consisting of a G-quadruplex(G4)-modulated tumor-targeted DNA vehicle and a well-designed cellular stimuli-responsive ligand-drug conjugates (LDCs) is proposed. An original "donor-acceptor" binary fluorescent ligand, with ultrahigh affinity, brightness, and photostability, is engineered to tightly bind G4 structures and significantly improve the nuclease resistance of the DNA vehicle, which serves as a bridge contributing to the construction of the prodrug system, named ApG4/LDCs. Sodium nitroprusside and doxorubicin are loaded into ApG4/LDCs in one pot and generate nitric oxide and superoxide anion in response to cancer cellular environments, which in cascade generates peroxynitrite to cause DNA damage while promoting the self-monitored drug release to achieve enhanced targeted therapy. Such a cascade activation and self-reinforcement process is executed only when the prodrug system targets the tumor tissue followed by cell uptake, showing significant antitumor efficacy and greatly weakening the damage to normal tissues. Given the unique features, the innovative strategy for prodrug design may open a new door to precision disease treatment.

G-Quadruplex-Programmed Versatile Nanorobot Combined with Chemotherapy and Gene Therapy for Synergistic Targeted Therapy.

Developing synergistic targeted therapeutics to improve treatment efficacy while reducing side effects has proven promising for anticancer therapies, but how to conveniently modulate multidrug cooperation remains a challenge. Here, a novel synergistic strategy using a G-quadruplex-programmed versatile nanorobot (G4VN) containing two subunits of DNAzyme (DzG4) and ligand-drug conjugates (LDCs) is proposed to precisely target tumors and then execute both gene silencing and chemotherapy. As the core module of this nanorobot, a well-designed G4 responding to a high level of K+ in tumor microenvironment smartly kills three birds with one stone, which makes two TfR aptamers proximate to improve their efficiency of targeting tumor cells, and in situ activates a split 10-23 DNAzyme to downregulate target mRNA expression, meanwhile promotes the cell uptake of a GSH-responsive LDCs to enhance drug efficacy. Such a design enables a potently synergistic anticancer therapy with low side effects in vivo, showing great promise for broad applications in precision disease treatment.

Bispecific Nanobody-Aptamer Conjugates for Enhanced Cancer Therapy in Solid Tumors.

Bispecific antibodies possess exceptional potential as therapeutic agents due to their capacity to bind to two different antigens simultaneously. However, challenges pertain to unsatisfactory stability, manufacturing complexity, and limited tumor penetration hinder their broad applicability. In this study, a versatile technology is presented for the rapid generation of bispecific nanobody-aptamer conjugates with efficient tumor penetration. The approach utilizes microbial transglutaminase (MTGase) and click chemistry to achieve site-specific conjugation of nanobodies and aptamers, which are termed nanotamers. The nanotamers recognize and bind to two types of molecular targets expressed on cancer cells. As a prototype, a bispecific nanotamer is developed that binds both clusters of differentiation 47 (CD47) and mesenchymal epithelial transition receptor (Met) expressed on the tumor cell membrane. This CD47-Met nanotamer demonstrates high affinity and specificity toward tumor cells expressing both targets, exhibits improved receptor functional inhibition through a strong steric hindrance effect. Moreover, its capacity for deep tumor penetration greatly enhances the impact of conventional chemotherapy on antitumor efficacy. The as-developed nanotamer synthesis approach shows promise to customize bispecific molecular probes targeting different cancer types and different therapeutic goals.

Drop-by-Drop Addition of Reagents to a Double Emulsion.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.