Aptamer-Based Multiparameter Analysis for Molecular Profiling of Hematological Malignancies.

The subtypes of hematological malignancies (HM) with minimal molecular profile differences display an extremely heterogeneous clinical course and a discrepant response to certain treatment regimens. Profiling the surface protein markers offers a potent solution for precision diagnosis of HM by differentiating among the subtypes of cancer cells. Herein, we report the use of Cell-SELEX technology to generate a panel of high-affinity aptamer probes that are able to discriminate subtle differences among surface protein profiles between different HM cells. Experimental results show that these aptamers with apparent dissociation constants (Kd) below 10 nM display a unique recognition pattern on different HM subtypes. By combining a machine learning model on the basis of partial least-squares discriminant analysis, 100% accuracy was achieved for the classification of different HM cells. Furthermore, we preliminarily validated the effectiveness of the aptamer-based multiparameter analysis strategy from a clinical perspective by accurately classifying complex clinical samples, thus providing a promising molecular tool for precise HM phenotyping.

Aptamer-Mediated Enrichment of Rare Circulating Fetal Nucleated Red Blood Cells for Noninvasive Prenatal Diagnosis.

Isolation of circulating fetal nucleated red blood cells (cfNRBCs) from maternal peripheral blood provides a superior strategy for noninvasive prenatal genetic diagnosis. Recent technical advances in single-cell isolation and genetic analyses have promoted the clinical application of circulating fetal cell-based noninvasive prenatal diagnosis. However, the lack of highly specific ligands for rare circulating fetal cell enrichment from massive maternal cells significantly impedes the clinical transformation progress. In this work, aptamers specific to NRBCs were developed through clinical sample-based cell-SELEX. Herein, the complex clinical system provides natural selection stringency through binding competition between target and background cells, and it empowers aptamers with high specificity. An aptamer-based strategy was also established to isolate cfNRBCs from maternal peripheral blood. Results show the remarkable selectivity and affinity of developed aptamers, enabling efficient enrichment of cfNRBCs from abundant maternal cells. Moreover, screening for fetal sex and trisomy syndrome achieved high accuracy through chromosome analysis of enriched cfNRBCs. To the best of our knowledge, this is the first report to develop aptamer ligands for cfNRBC enrichment, providing an efficient strategy to screen cfNRBC-specific ligands and demonstrating broad application potential for cfNRBC-based noninvasive prenatal diagnosis.

An Implantable Magnetic Vascular Scaffold for Circulating Tumor Cell Removal In Vivo.

An integrated trapped device (ITD) capable of removal of circulating tumor cells (CTCs) can assuage or even prevent metastasis. However, adhesion repertoires are ordinarily neglected in the design of ITDs, possibly leading to the omission of highly metastatic CTC and treatment failure. Here a vascular-like ITD with adhesive sites and wireless magnetothermal response to remove highly metastatic CTC in vivo is presented. Such a vascular-like ITD comprises circumferential well-aligned fibers and artificial adhesion repertoires and is optimized for magnetothermal integration. Continuous and repeated capture in a dynamic environment increases capture efficiency over time. Meanwhile, the heat generation of the ITD leads to the capture of CTC death owing to cell heat sensitivity. Furthermore, the constructed bioinspired ultrastructure of the ITD prevents vascular blockage and induces potential vascular regeneration. Overall, this work defines an extendable strategy for constructing adhesion repertoires against intravascular shear forces, provides a vascular-like ITD for reducing CTC counts, and is expected to alleviate the risk of cancer recurrence.

Development of a novel PROTAC using the nucleic acid aptamer as a targeting ligand for tumor selective degradation of nucleolin.

PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and are poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs.

Elucidation of CKAP4-remodeled cell mechanics in driving metastasis of bladder cancer through aptamer-based target discovery.

Metastasis contributes to the dismal prognosis of bladder cancer (BLCA). The mechanical status of the cell membrane is expected to mirror the ability of cell migration to promote cancer metastasis. However, the mechanical characteristics and underlying molecular profile associated with BLCA metastasis remain obscure. To study the unique cellular architecture and traits associated with cell migration, using a process called cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX) we generated an aptamer-based molecular probe, termed spl3c, which identified cytoskeleton-associated protein 4 (CKAP4). CKAP4 was associated with tumor metastasis in BLCA, but we also found it to be a mechanical regulator of BLCA cells through the maintenance of a central-to-peripheral gradient of stiffness on the cell membrane. Notably, such mechanical traits were transportable through exosome-mediated intercellular CKAP4 trafficking, leading to significant enhancement of migration in recipient cells and, consequently, aggravating metastatic potential in vivo. Taken together, our study shows the robustness of this aptamer-based molecular tool for biomarker discovery, revealing the dominance of a CKAP4-induced central-to-peripheral gradient of membrane stiffness that benefits cell migration and delineating the role of exosomes in mediating mechanical signaling in BLCA metastasis.

DNA-Based Molecular Engineering of the Cell Membrane.

The cell membrane serves as a barrier and gatekeeper to regulate the cellular transportation of substances and information. It plays a significant role in protecting the cell from the extracellular environment, maintaining intracellular homeostasis, and regulating cellular function and behaviors. The capability to engineer the cell membrane with functional modules that enable dynamic monitoring and manipulating the cell-surface microenvironment would be critical for studying molecular mechanisms underlying various biological processes. To meet this goal, DNA, with intrinsic advantages of high versatility, programmability, and biocompatibility, has gained intense attention as a molecular tool for cell-surface engineering. The past three decades have witnessed the rapid advances of diverse nucleic acid materials, including functional nucleic acids (FNAs), dynamic DNA circuits, and exquisite DNA nanostructures. In this mini review, we have summarized the recent progress of DNA technology for cell membrane engineering, particularly focused on their applications for molecular sensing and imaging, precise cell identification, receptor activity regulation, and artificial membrane structures. Furthermore, we discussed the challenge and outlook on using nucleic acid materials in this specific research area.

Nucleic acid aptamer controls mycoplasma infection for inhibiting the malignancy of esophageal squamous cell carcinoma.

Esophageal cancer is one of the most frequent malignant tumors of the digestive tract, among which esophageal squamous cell carcinoma (ESCC) is the main pathological type worldwide. Previous studies have shown microbial infections in the upper digestive tract to be a potential risk factor in ESCC etiology. In this study, we identified that Mycoplasma hyorhinis infection promoted the malignancy of ESCC. In response, we generated a single-stranded DNA aptamer, ZY3A, against M. hyorhinis using the cell-SELEX strategy. The underlying recognition mechanism of ZY3A on M. hyorhinis involves its binding to M. hyorhinis-specific p37 protein. This tool allowed us to provide the first proof-of-concept evidence using a nucleic acid aptamer to control mycoplasma infection. More specifically, we found that ZY3A could neutralize M. hyorhinis infection on ESCC cells by blocking the interaction between p37 protein and its receptor TLR4 on the ESCC cell membrane. As a result, ZY3A inhibited the migration and invasion of M. hyorhinis-infected ESCC cells in vitro and metastasis in vivo. Taken together, these findings indicate that aptamer ZY3A is a potential candidate for development into a novel molecular tool for treatment of M. hyorhinis infection and a safe first-in-class M. hyorhinis-targeting antitumor agent.

A Dual-Targeting Circular Aptamer Strategy Enables the Recognition of Different Leukemia Cells with Enhanced Binding Ability.

Currently, the broad use of monovalent aptamers in oncology faces challenges, including insufficient recognition and internalization caused by a finite number of receptors on the cell surface, as well as a confined recognition spectrum. Herein, we describe the development of a dual-targeting circular aptamer (DTCA) that can recognize two different biomarkers on living cells to augment aptamer-receptor interactions, thus enhancing recognition of the target cells. This improvement not only boosts binding and internalization abilities, but also expands the recognition spectrum of these aptamers to different leukemia cells. Moreover, the stability of DTCA in serum can be significantly improved by an enzyme-promoted terminal ligation strategy. The chemical incorporation of 5-fluorodeoxyuridine into DTCA resulted in a pharmaceutically functional aptamer that exhibited excellent selectivity, as demonstrated by its high cytotoxicity against target cancer cells, but not to normal cells. The superiority of our newly developed strategy was further highlighted by its precise tumor-imaging capability.

Development of a DNA Aptamer against Multidrug-Resistant Hepatocellular Carcinoma for In Vivo Imaging.

Hepatocellular carcinoma (HCC) is a type of cancer that has high rates of recurrence and mortality. One of the most important factors that lead to treatment failure of HCC is the acquisition of multidrug resistance (MDR). Development of specific ligands for multidrug-resistant HCC will provide useful molecular tools for precise diagnosis and targeted theranostics. Herein, a multidrug-resistant HCC cell (HepG2/MDR)-specific aptamer was developed through Cell-SELEX (systematic evolution of ligands by exponential enrichment) technology. With dissociation constants lying in the nanomolar range, the molecularly designed PS-ZL-7c aptamer showed great selectivity to drug-resistant cancer cells. The in vivo imaging results illustrated that the PS-ZL-7c specifically accumulated in the drug-resistant tumors but not in drug-sensitive tumors and normal tissues, indicating that the PS-ZL-7c aptamer possessed excellent potential as a targeting ligand for precise diagnosis and target theranostics of multidrug-resistant HCC.

The regulation of NONO by USP11 via deubiquitination is linked to the proliferation of melanoma cells.

Ubiquitin-specific protease 11 (USP11) has been implicated in the regulation of DNA repair, apoptosis, signal transduction and cell cycle. It belongs to a USP subfamily of deubiquitinases. Although previous research has shown that USP11 overexpression is frequently found in melanoma and is correlated with a poor prognosis, the potential molecular mechanism of USP11 in melanoma remains indefinitive. Here, we report that USP11 and NONO colocalize and interact with each other in the nucleus of melanoma cells. As a result, the knockdown of USP11 decreases NONO levels. Whereas, overexpression of USP11 increases NONO levels in a dose-dependent manner. Furthermore, we reveal that USP11 protects NONO protein from proteasome-mediated degradation by removing poly-ubiquitin chains conjugated onto NONO. Functionally, USP11 mediated melanoma cell proliferation via the regulation of NONO levels because ablation of USP11 inhibits the proliferation which could be rescued by ectopic expression of NONO protein. Moreover, a significant positive correlation between USP11 and NONO concentrations was found in clinical melanoma samples. Collectively, these results demonstrate that USP11 is a new deubiquitinase of NONO and that the signalling axis of USP11-NONO is significantly involved in melanoma proliferation.

Meta-DNA structures.

DNA origami has emerged as a highly programmable method to construct customized objects and functional devices in the 10-100 nm scale. Scaling up the size of the DNA origami would enable many potential applications, which include metamaterial construction and surface-based biophysical assays. Here we demonstrate that a six-helix bundle DNA origami nanostructure in the submicrometre scale (meta-DNA) could be used as a magnified analogue of single-stranded DNA, and that two meta-DNAs that contain complementary 'meta-base pairs' can form double helices with programmed handedness and helical pitches. By mimicking the molecular behaviours of DNA strands and their assembly strategies, we used meta-DNA building blocks to form diverse and complex structures on the micrometre scale. Using meta-DNA building blocks, we constructed a series of DNA architectures on a submicrometre-to-micrometre scale, which include meta-multi-arm junctions, three-dimensional (3D) polyhedrons, and various 2D/3D lattices. We also demonstrated a hierarchical strand-displacement reaction on meta-DNA to transfer the dynamic features of DNA into the meta-DNA. This meta-DNA self-assembly concept may transform the microscopic world of structural DNA nanotechnology.

DNA-Modulated Plasmon Resonance: Methods and Optical Applications.

The near-field effects in the vicinity of metallic nanoparticle surfaces, as induced by electromagnetic radiation with specific wavelength, give rise to a variety of novel optical properties and attractive applications because of surface plasmons, which are the coherent oscillations of conduction electrons on a metal surface. The interdisciplinary field of plasmonics has witnessed vigorous growth, promoting research on the modulation of plasmon resonance by constructing advanced plasmonic nanoarchitectures with controllable size, morphology, or interparticle coupling. Among diversified tools, deoxyribonucleic nucleic acid (DNA) possesses prominent superiority as a result of its designability, programmability, addressability, and ease of nanomaterial modification. In this review, we focus on the methods and optical applications of plasmon resonance modulation accomplished by DNA nanotechnology. Recent developments in the construction of DNA-mediated plasmonic nanoarchitecture and key ongoing research directions utilizing unique optical features are highlighted. Obstacles and challenges in this field are pointed out, followed by preliminary suggestions on some areas of opportunity that deserve attention.

Single-Molecular Catalysis Identifying Activation Energy of the Intermediate Product and Rate-Limiting Step in Plasmonic Photocatalysis.

Plasmon-mediated photocatalysis provides a novel strategy for harvesting solar energy. Identification of the rate-determining step and its activation energy in plasmon-mediated photocatalysis plays critical roles for understanding the contribution of hot carriers, which facilitates rational designation of catalysts with integrated high photochemical conversion efficiency and catalytic performance. However, it remains a challenge due to a lack of research tools with spatiotemporal resolution that are capable of capturing intermediates. In this work, we used a single-molecule fluorescence approach to investigate a localized surface plasmon resonance (LSPR)-enhanced photocatalytic reaction with subturnover resolution. By introducing variable temperature as an independent parameter in plasmonic photocatalysis, the activation energies of tandem reaction steps, including intermediate generation, product generation, and product desorption, were clearly differentiated, and intermediate generation was found to be the rate-limiting step. Remarkably, the cause of the plasmon-enhanced catalysis performance was found to be its ability of lowering the activation energy of intermediate generation. This study gives new insight into the photochemical energy conversion pathways in plasmon-enhanced photocatalysis and sheds light on designing high-performance plasmonic catalysts.

Molecularly Engineering Triptolide with Aptamers for High Specificity and Cytotoxicity for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks three important receptors, ER, PR, and HER2. It is more aggressive and more likely to relapse after treatment, thus has been identified as one of the most malignant breast cancer types. The development of efficient targeted TNBC therapy is an important research topic in TNBC treatment. We report the development of a new aptamer-drug conjugate (ApDC), AS1411-triptolide conjugate (ATC), as targeted therapy for the treatment of TNBC with high efficacy. The conjugate possesses excellent specificity and high cytotoxicity against the MDA-MB-231 cell line. The advantages of our newly invented ATC are further highlighted by its excellent in vivo anti-TNBC efficacy and negligible side effects toward healthy organs.

Conjugating Aptamer and Mitomycin C with Reductant-Responsive Linker Leading to Synergistically Enhanced Anticancer Effect.

Mitomycin C (MMC) has been using for the treatment of a variety of digestive tract cancers. However, its nonspecific DNA-alkylating ability usually causes severe side effects, thus largely limiting its clinical applications. The utilization of an efficient active targeted drug delivery technique would address this issue. Accordingly, we report the design and development of aptamer-mitomycin C conjugates that use different cross-linking chemistry. The targeted delivery ability and cytotoxicity of these conjugates were carefully studied. It is worth noting that a linker-dependent cytotoxicity effect was observed for these conjugates. The use of a reductant-sensitive disulfide bond cross-linking strategy resulted in significantly enhanced cytotoxicity of MMC against the target cancer cell lines. Importantly, this cytotoxicity enhancement was suited to different types of aptamers, demonstrating the success of our design. Mechanistic studies of the enhanced cytotoxicity effect indicated that the target recognition, specific binding, and receptor-mediated internalization of aptamer were also critical for the observed effect.

Polymeric Engineering of Aptamer-Drug Conjugates for Targeted Cancer Therapy.

Nucleic acid aptamers, also known as "chemical antibodies", have been widely employed in targeted cancer therapy and diagnosis. For example, aptamer-drug conjugates (ApDCs), through covalent conjugation of cytotoxic warheads to aptamers, have demonstrated anticancer efficacy both in vitro and in vivo. However, a general strategy to endow ApDCs with enhanced biostability, prolonged circulation half-life, and high drug loading content remained elusive. Herein, we present a polymeric approach to engineer ApDCs via conjugation of cell-targeting aptamers with water-soluble polyprodrugs containing a reductive environmentally sensitive prodrug and biocompatible brush-like backbone. The resultant high-drug loading Aptamer-PolyproDrug Conjugates (ApPDCs) exhibited high nuclease resistance, extended in vivo circulation time, specific recognition, and cellular uptake to target cells, reduction-triggered and fluorescent-reporting drug release, and effective cytotoxicity. We could also further expand this design principle toward combination therapy by using two kinds of therapeutic drugs with distinct pharmacological mechanisms.

A basic insight into aptamer-drug conjugates (ApDCs).

Aptamers are often compared with antibodies since both types of molecules function as targeting ligands for specific cancer cell recognition. However, aptamers offer several advantages, including small size, facile chemical modification, high chemical stability, low immunogenicity, rapid tissue penetration, and engineering simplicity. Despite these advantages, several crucial factors have delayed their clinical translation, such as concerns over inherent physicochemical stability and safety. Meanwhile, steps have been taken to make aptamer-drug conjugates, or ApDCs, a clinically practical tool. In this review, we highlight the development of ApDCs and discuss how researchers are solving some problems associated with their clinical application for targeted therapy.

Reactivating Catalytic Surface: Insights into the Role of Hot Holes in Plasmonic Catalysis.

Surface plasmon resonance of coinage metal nanoparticles is extensively exploited to promote catalytic reactions via harvesting solar energy. Previous efforts on elucidating the mechanisms of enhanced catalysis are devoted to hot electron-induced photothermal conversion and direct charge transfer to the adsorbed reactants. However, little attention is paid to roles of hot holes that are generated concomitantly with hot electrons. In this work, 13 nm spherical Au nanoparticles with small absorption cross-section are employed to catalyze a well-studied glucose oxidation reaction. Density functional theory calculation and X-ray absorption spectrum analysis reveal that hot holes energetically favor transferring catalytic intermediates to product molecules and then desorbing from the surface of plasmonic catalysts, resulting in the recovery of their catalytic activities. The studies shed new light on the use of the synergy of hot holes and hot electrons for plasmon-promoted catalysis.

Multicolor Gold-Silver Nano-Mushrooms as Ready-to-Use SERS Probes for Ultrasensitive and Multiplex DNA/miRNA Detection.

Uniform silver-containing metal nanostructures with strong and stable surface-enhanced Raman scattering (SERS) signals hold great promise for developing ultrasensitive probes for biodetection. Nevertheless, the direct synthesis of such ready-to-use nanoprobes remains extremely challenging. Herein we report a DNA-mediated gold-silver nanomushroom with interior nanogaps directly synthesized and used for multiplex and simultaneous SERS detection of various DNA and RNA targets. The DNA involved in the nanostructures can act as not only gap DNA (mediated DNA) but also probe DNA (hybridized DNA), and DNA's involvement enables the nanostructures to have the inherent ability to recognize DNA and RNA targets. Importantly, we were the first to establish a new method for the generation of multicolor SERS probes using two different strategies. First Raman-labeled alkanethiol probe DNA was assembled on gold nanoparticles, and second, thiol-containing Raman reporters were coassembled with the probe DNA. The ready-to-use probes also give great potential to develop ultrasensitive detection methods for various biological molecules.