PAMless SpRY exhibits a preference for the seed region for efficient targeting.

Protospacer-adjacent motif (PAM) recognition licenses Cas nucleases for genome engineering applications, thereby restricting gene targeting to PAM-containing regions. Protein engineering has led to PAM-relaxed SpCas9 variants like SpG and SpRY. Given the evolved role of PAMs in facilitating target-searching kinetics, it remains unclear how these variants quickly locate their targets. We show that SpG and SpRY exhibit a preference for the seed region. To compensate for the relaxed PAM recognition, SpRY has evolved a sequence preference for the seed region through interactions with A61R and A1322R. Furthermore, SpCas9 exhibits a significant decrease in target search kinetics on high-PAM-density DNA, slowing down up to three orders of magnitude compared to low-PAM-density DNA, suggesting the necessity for sequence recognition even in PAM-relaxed variants. This underscores the importance of considering Cas9 target-searching kinetics in SpCas9 PAMless engineering, providing valuable insights for further PAMless Cas9 protein engineering efforts.

Hierarchical integration of DNA nanostructures and NanoGold onto a microchip facilitates covalent chemistry-mediated purification of circulating tumor cells in head and neck squamous cell carcinoma.

It is well-established that the combined use of nanostructured substrates and immunoaffinity agents can enhance the cell-capture performance of the substrates, thus offering a practical solution to effectively capture circulating tumor cells (CTCs) in peripheral blood. Developing along this strategy, this study first demonstrated a top-down approach for the fabrication of tetrahedral DNA nanostructure (TDN)-NanoGold substrates through the hierarchical integration of three functional constituents at various length-scales: a macroscale glass slide, sub-microscale self-organized NanoGold, and nanoscale self-assembled TDN. The TDN-NanoGold substrates were then assembled with microfluidic chaotic mixers to give TDN-NanoGold Click Chips. In conjunction with the use of copper (Cu)-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated CTC capture and restriction enzyme-triggered CTC release, TDN-NanoGold Click Chips allow for effective enumeration and purification of CTCs with intact cell morphologies and preserved molecular integrity. To evaluate the clinical utility of TDN-NanoGold Click Chips, we used these devices to isolate and purify CTCs from patients with human papillomavirus (HPV)-positive (+) head and neck squamous cell carcinoma (HNSCC). The purified HPV(+) HNSCC CTCs were then subjected to RT-ddPCR testing, allowing for detection of E6/E7 oncogenes, the characteristic molecular signatures of HPV(+) HNSCC. We found that the resulting HPV(+) HNSCC CTC counts and E6/E7 transcript copy numbers are correlated with the treatment responses in the patients, suggesting the potential clinical utility of TDN-NanoGold Click Chips for non-invasive diagnostic applications of HPV(+) HNSCC.

Size-Controllable DNA Origami-Stacked Gold Nanoparticles for Deep Tumor-Penetrating Therapy.

With the rapid development of nanotechnology, researchers have designed a variety of intelligent nanodelivery systems to enhance tumor targeting of anticancer drugs. However, increased tumor accumulation does not indicate deeper penetration in the tumor tissue, without which the tumor cells in the core area cannot be sufficiently killed. Herein, we develop a size-controllable nanoparticle system for deep-penetrating cancer therapy, which will be programmably disassembled with the decrease of the pH from the normal tissue to the tumor microenvironment and to the intracellular area. The integrated nanoparticle is composed of a gold nanoparticle (GNP, ∼30 nm) and a tetrahedral DNA nanostructure (TDN, ∼25 nm) loaded with doxorubicin (DOX). Initially, the nanoparticles maintain a larger size (∼100 nm) to accumulate in the tumor through the enhanced permeability and retention effect. At a pH of about 6.5 at the tumor microenvironment, with the linkage of DNA sequences converting into a triplex structure, the TDNs detach from the GNP and penetrate deeply into the tumor interstitium and then are internalized into the cells. Finally, in acidic lysosomes with pH 5.0, the TDNs release DOX by forming an i-motif structure. This nanosmart delivery system thus shows effective deep penetration into the tumor core with good antitumor efficacy and satisfactory biocompatibility and provides new insights into the development of intelligent nanosystems for anti-cancer treatment.

A novel electrochemical aptasensor for exosomes determination and release based on specific host-guest interactions between cucurbit [7]uril and ferrocene.

The superior supramolecular recognition ability of macrocyclic compounds will enhance the sensitivity and selectivity of electrochemical detection, which has a great application potential in electrochemical sensing. Herein, we developed a novel electrochemical aptasensor based on the specific host-guest interactions between cucurbit [7]uril and ferrocene (Fc) for capture, determination and release of exosomes. Macrocyclic compounds, cucurbit [7]uril is modified on the surface of the gold nanoparticles composed electrode by self-assembling. CD63 aptamer linked ferrocene is introduced into this platform to capture exosomes specifically by CD63 protein on the exosomes. The dual specificity of macrocyclic compounds and aptamers enables highly selective and sensitive electrochemical detection of exosomes. The limit of detection (LOD) was 482 particles µL-1. In addition, the captured exosomes could be released on demand in a very mild manner through aminoferrocene (NH2-Fc) because of its higher affinity to cucurbit [7]uril. The proposed electrochemical aptasensor showed good performance in detecting exosomes even in plasma samples, thus demonstrating its great potential in early clinical diagnosis. Simultaneously, exosomes could be released undamaged by this protocol, exhibiting good applicability in comprehensive studies of exosomes. Moreover, this strategy can be applied to other target biomolecules by changing the recognition pairs.

DNA-Based pH Nanosensor with Adjustable FRET Responses to Track Lysosomes and pH Fluctuations.

Extensive attention has been recently focused on designing signal adjustable biosensors. However, there are limited approaches available in this field. In this work, to visually track lysosomes with high contrast, we used the i-motif structure as a pH-responsive unit and proposed a novel strategy to regulate the fluorescence resonance energy transfer (FRET) response of the pH sensor. By simply splitting the i-motif into two parts and modulating the split parameters, we can tune the pH transition midpoint (pHt) from 5.71 to 6.81 and the signal-to-noise ratio (S/N) from 1.94 to 18.11. To facilitate the lysosome tracking, we combined the i-motif split design with tetrahedral DNA (Td). The obtained pH nanosensor (pH-Td) displays appropriate pHt (6.12) to trace lysosomes with high S/N (10.3). Benefited from the improved stability, the superior cell uptake and lysosomal location of pH-Td, the visualization of the distribution of lysosomes, the lysosome-mitochondria interaction, and the pH changes of lysosomes in response to different stimuli were successfully achieved in NIH 3T3 cells. We believe that the design concept of controlling the split sequence distance will provide a novel insight into the design of i-motif-based nanosensors and even inspire the construction of smart DNA nanodevices for sensing, disease diagnosis, and controllable drug delivery.

Synthesis of Diboronic Acid-Based Fluorescent Probes for the Sensitive Detection of Glucose in Aqueous Media and Biological Matrices.

Reliable and accurate glucose detection in biological samples is of great importance in clinical diagnosis and medical research. Chemical probes are advantageous in simple operation and flexible design, especially for the development of fluorescent probes. Anthracene-based diboronic acid (P-DBA) has shown potential in glucose probing because of its high sensitivity. However, poor solubility limits its applications in aqueous media. In this work, we systemically modify P-DBA by introducing fluoro (F-), chloro (Cl-), methoxyl (MeO-), or cyano (CN-) substituents. Among these probes, the cyano-substituted probe (CN-DBA) displays the highest glucose-binding constant (6489.5 M-1, 33% MeOH). More importantly, it shows good water solubility in the aqueous solution (0.5% MeOH), with ultrasensitive recognition with glucose (LOD = 1.51 µM) and robust sensing from pH 6.0 to 9.0. Based on these features, the CN-DBA is finally applied to detect glucose in cell lysates and plasma, with satisfactory recovery and precision. These results demonstrate that CN-DBA could serve as an accurate, sensitive fluorescent probe for glucose assays in biological samples.

Improved cancer phototheranostic efficacy of hydrophobic IR780 via parenteral route by association with tetrahedral nanostructured DNA.

As a photosensitizer with effective photothermal (PTT) and photodynamic (PDT) response, IR780 has been widely explored as promising cancer phototheranostic molecule. However, the systematic administration of IR780 usually suffers from poor water solubility and low photostability, so that it cannot be administrated by parenteral route. In this study, we design a tetrahedral DNA (Td)-based nanosystem to load IR780 (IR780@Td) via electrostatic interaction and π-π stacking. After encapsulation, the water solubility and photostability of IR780 have been greatly improved, and the IR780@Td shows an appropriate nanoformulated size (224 nm) to facilitate hyperthermia-mediated tumor targeting by EPR effect. The nanostructure of Td is proved to be crucial for the proper size and good stability of IR780@Td nanoformulation for in vivo application. The in vitro and ex vivo PTT/PDT efficiencies of IR780 are improved in IR780@Td group. In the tumor-bearing mice, the accumulation of IR780 in tumor site is significantly high in IR780@Td group. Under near-infrared laser irradiation, the intravenous administration of IR780@Td promotes the tumor imaging and enhances anti-tumor effect than IR780 treatment. In summary, the proposed strategy shows promising effect in facilitating intravenous injection of IR780 and enhancing the phototheranostic efficacy for cancer treatment.

AIEgens Conjugation Improves the Photothermal Efficacy and Near-Infrared Imaging of Heptamethine Cyanine IR-780.

Near-infrared (NIR) fluorescent probes can deeply penetrate through tissues with little damage. To facilitate image-guided theranostics, researchers usually apply a desired amount of photosensitizers to achieve effective photothermal responses. However, these probes could easily suffer from low photostability and aggregated-caused quenching effect in high concentrations. In this paper, the rational incorporation of an aggregated-induced emission (AIE) unit into the structure of heptamethine cyanine IR-780 is reported. Using tetraphenylethene (TPE) as an AIE core, we synthesize three TPE-modified IR-780 probes (IR-780 AIEgens) via different linkages. The IR-780 derivatives all show enhanced AIE features, in which the probe with an ether linkage (IR780-O-TPE) is superior in rapid cell uptake, high targeting capacity, and good photostability. Moreover, IR780-O-TPE exhibits the strongest cytotoxicity to HeLa cells (IC50 = 3.3 µM). The three IR-780 derivatives displayed a photothermal response in a concentration-dependent manner, in which IR-780 AIEgens are more cytotoxic than IR-780, with IC50 of 0.3 µM under 808 nm laser irradiation. In tumor-bearing mice, the optimal probe IR780-O-TPE also showed a more effective photothermal response than IR-780. By illustrating the relationship between aggregation state with photophysical properties, cell imaging, and cytotoxicity, this work is helpful in modulating NIR-based photosensitizers into AIE features for efficient image-guided theranostics.

High-Efficiency Isolation and Rapid Identification of Heterogeneous Circulating Tumor Cells (CTCs) Using Dual-Antibody-Modified Fluorescent-Magnetic Nanoparticles.

Extreme rarity and inherent heterogeneity of circulating tumor cells (CTCs) result in a tremendous challenge for the CTC isolation from patient blood samples with high efficiency and purity. Current CTC isolation approaches mainly rely on the epithelial cell adhesion molecule (EpCAM), which may significantly reduce the ability to capture CTCs when the expression of EpCAM is lost or down-regulated in epithelial-mesenchymal transition. Here, a rapid and highly efficient method is developed to isolate and identify heterogeneous CTCs with high efficiency from patient blood samples using the fluorescent-magnetic nanoparticles (F-MNPs). A dual-antibody interface targeting EpCAM and N-cadherin is fabricated onto the F-MNPs to capture epithelial CTCs as well as mesenchymal CTCs from whole blood samples. The poly(carboxybetaine methacrylate) brushes of excellent antifouling properties are employed to decrease nonspecific cell adhesion. Moreover, the F-MNPs provide a prompt identification strategy for heterogeneous CTCs (F-MNPs+, Hoechst 33342+, and CD45-) that can directly identify CTCs in a gentle one-step processing within 1 h after isolation from patient blood samples. This has been demonstrated through artificial samples as well as patient samples in details.