Recent advance in dual-functional luminescent probes for reactive species and common biological ions.

The chemical microenvironment in cells is extremely complex involving numerous species with drastically different concentration as well as temporal and spatial distribution. Especially, reactive species including ROS, RNS, RSS, and many inorganic ions are recognized to play very important roles. The concentrations of these species are constantly regulated and often depend on each other. While fluorescent probes have been widely used to study various cell biological processes, those simultaneously probing two species just emerged recently. In this review, we highlight dual-functional luminescent probes for the detection and fluorescence imaging of two and more analytes in cells. The content will cover small-molecule synthetic fluorescent probes, DNA nanoassemblies, and nanoparticle-based nanoprobes. The target analytes include reactive species such as ROS, RNS, and RSS and other ions and molecules such as H+, Cl-, Ca2+, Cu2+, and O2.

A Chimeric Conjugate of Antibody and Programmable DNA Nanoassembly Smartly Activates T Cells for Precise Cancer Cell Targeting.

Synthetically directing T-cells against tumors emerges as a promising strategy in immunotherapy, while it remains challenging to smartly engage T cells with tunable immune response. Herein, we report an intelligent molecular platform to engineer T-cell recognition for selective activation to potently kill cancer cells. To this end, we fabricated a hybrid conjugate that uses a click-type DNA-protein conjugation to equip the T cell-engaging antibody with two distinct programmable DNA nanoassemblies. By integrating multiple aptameric antigen-recognitions within a dynamic DNA circuit, we achieved combinatorial recognition of triple-antigens on cancer cells for selective T-cell activation after high-order logic operation. Moreover, by coupling a DNA nanostructure, we precisely defined the valence of the antigen-binding aptamers to tune avidity, realizing effective tumor elimination in vitro and in vivo. Together, we present a versatile and programmable strategy for synthetic immunotherapy.

Amplified fluorescence imaging of HER2 dimerization on cancer cells by using a co-localization triggered DNA nanoassembly.

Convenient and sensitive detection of human epidermal growth factor receptor 2 (HER2) dimerization is highly desirable for molecule subtyping and guiding personalized HER2 targeted therapy of breast cancer. A colocalization-triggered DNA nanoassembly (CtDNA) strategy was developed for amplified imaging of HER2 dimerization. It exploits (a) the advantage of the specificity of aptamer proximity hybridization, and (b) the high sensitivity of hairpin-free nonlinear HCR. The mechanism of step-by-step hairpin-free nonlinear HCR for DNA dendritic nanoassembly was studied by native polyacrylamide gel electrophoresis, atomic force microscopy and fluorometry. The results revealed a high specificity, sensitivity, and excellent controllability of the DNA dendritic nanoassembly. The method was used to identify HER2 homodimers and HER2/HER3 heterodimers in various breast cancer cell lines using fluorescence microscopy. It was then extended to image and quantitatively evaluate HER2 homodimers in clinical formalin-fixed paraffin-embedded breast cancer tissue specimens. This revealed its remarkable accuracy and practicality for clinical diagnostics. Graphical abstract Schematic presentation of amplified imaging of human epidermal growth factor receptor 2 (HER2) dimerization on cancer cell surfaces by using a co-localization triggered DNA nanoassembly (CtDNA).

DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA.

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL-1, 1.5 copies⋅µL-1, and 0.17 copies⋅µL-1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

Bioorthogonal Microbubbles with Antifouling Nanofilm for Instant and Suspended Enrichment of Circulating Tumor Cells.

Integrating clinical rare cell enrichment, culture, and single-cell phenotypic profiling is currently hampered by the lack of competent technologies, which typically suffer from weak cell-interface collision affinity, strong nonspecific adsorption, and the potential uptake. Here, we report cells-on-a-bubble, a bioinspired, self-powered bioorthogonal microbubble (click bubble) that leverages a clickable antifouling nanointerface and a DNA-assembled sucker-like polyvalent cell surface, to enable instant and suspended isolation of circulating tumor cells (CTCs) within minutes. Using this biomimetic engineering strategy, click bubbles achieve a capture efficiency of up to 98%, improved by 20% at 15 times faster over their monovalent counterparts. Further, the buoyancy-activated bubble facilitates self-separation, 3D suspension culture, and in situ phenotyping of the captured single cancer cells. By using a multiantibody design, this fast, affordable micromotor-like click bubble enables suspended enrichment of CTCs in a cohort (n = 42) across three cancer types and treatment response evaluation, signifying its great potential to enable single-cell analysis and 3D organoid culture.

Mechanosensing view of SARS-CoV-2 infection by a DNA nano-assembly.

The mechanical force between a virus and its host cell plays a critical role in viral infection. However, characterization of the virus-cell mechanical force at the whole-virus level remains a challenge. Herein, we develop a platform in which the virus is anchored with multivalence-controlled aptamers to achieve transfer of the virus-cell mechanical force to a DNA tension gauge tether (Virus-TGT). When the TGT is ruptured, the complex of binding module-virus-cell is detached from the substrate, accompanied by decreased host cell-substrate adhesion, thus revealing the mechanical force between whole-virus and cell. Using Virus-TGT, direct evidence about the biomechanical force between SARS-CoV-2 and the host cell is obtained. The relative mechanical force gap (<10 pN) at the cellular level between the wild-type virus to cell and a variant virus to cell is measured, suggesting a possible positive correlation between virus-cell mechanical force and infectivity. Overall, this strategy provides a new perspective to probe the SARS-CoV-2 mechanical force.

Self-assembly of a polythymine embedded activatable molecular beacon for one-step quantification of terminal deoxynucleotidyl transferase activity.

We describe an isothermal, single-reaction, and one-step method for signal-on quantification of terminal deoxynucleotidyl transferase (TdT) activity based on the periodic elongation and assembly of polythymine embedded activatable molecular beacon (PTA-MB) into DNA nanostructures. PTA-MB is easily designed according to the rule of the conventional molecular beacon (MB) but engineered with a polyT composed loop. Upon exposure to the specific target TdT, the MB is first elongated with an adenine-rich (A-rich) long chain so that it can then act as the anchoring substrate to capture many original PTA-MBs along its strand. Their unfolding contributes to preliminary fluorescence emission. Significantly, the assembled PTA-MBs can also be elongated and hybridized with residual free PTA-MBs for the second round of signal amplification. Accordingly, multiple rounds of elongation, assembly, and activation of initial PTA-MBs can lead to the formation of DNA nanostructures and induce a dramatically enhanced fluorescence signal for qualitative and quantitative evaluation of TdT activity. The final assay indicated a limit of detection (LOD) of 0.042 U mL-1 TdT and showed excellent selectivity for TdT versus other common enzymes. Moreover, the practical applicability was validated by direct/absolute quantification of TdT in real biological specimens and accurate monitoring of the activity of TdT pretreated by low/high temperature and heavy metal ions. These findings demonstrated that this functional PTA-MB and its unique assembly behavior is most likely to promote the study of oligonucleotide probe-based DNA assembly, providing a reliable, convenient, and universal platform for precise and point-of-care monitoring of various biomolecules.

Stepwise nanoassembly of a single hairpin probe and its biosensing.

Herein, we describe a novel trigger-induced DNA nanoassembly method using only one loop-stem shaped hairpin probe (HP) that consists of three different functional regions as a single building unit. The Region I is designed complementary to the trigger, while the Region II and Region III are projected to complementary with each other. When hybridized with the trigger, a toehold mediated strand displacement (TMSD) occurred on the strand of Region I, leading to the release of Region III for further hybridization with the Region II on another HP molecule and in turn inducing a stepwise growth of HP with the aid of polymerase. Unlike the conventional assembly approaches that rely on the sophisticated sequence design and complex operation, the single-HP nanoassembly is easy and fast. Moreover, because many HPs are opened during the assembly process, we exemplified the nanoassembly strategy by re-designing a new labeled hairpin probe to analyze the Kras oncogene with a high sensitivity and specificity. The present study demonstrated a novel promising DNA nanoassembly strategy for biological applications.

Programmable nanoassembly consisting of two hairpin-DNAs for p53 gene determination.

As one of the most exciting building blocks, DNA has gained increasing attention in the construction of promising nanostructures for various biological and medical purposes. In this contribution, we have developed an easily constructed DNA nanoassembly-based biosensing system that consists of one signal hairpin probe (SHP) and one label-free hairpin probe (LHP) for target p53 gene analysis. The probes of SHP and LHP were designed to be incapable of interacting with each other in the absence of the p53 gene. When the target gene is introduced, the 3' end of SHP (or LHP) hybridizes with the middle region of LHP (or SHP), leading to polymerase-sustained DNA nanoassembly. Because one target species can exhaust many building scaffolds to execute the programmable nanoassembly in one-pot approach, the fluorescence intensity of SHP is greatly enhanced in the presence of target gene in a simple and robust manner. The practical applicability was successfully demonstrated by screening target gene extracted from cancer cells. We believe this intriguing sensing strategy and desirable assay ability would provide new opportunities to develop versatile biochemical analysis methods.

Target-triggered DNA nanoassembly on quantum dots and DNAzyme-modulated double quenching for ultrasensitive microRNA biosensing.

Herein, a simple and novel fluorescence biosensing strategy has been developed for ultrasensitive determination of microRNA (miRNA) by combining target-triggered DNA nanoassembly on quantum dots (QDs) with DNAzyme-modulated double quenching of QDs. In presence of miRNA target, the target triggered catalytic hairpin assembly (CHA) amplification and powered highly efficient DNA nanoassembly on the surface of QDs, leading to exhibition of numerous G-quadruplexes close to the QDs. The G-quadruplex folded properly and bound hemin to form a stable G-quadruplex/hemin complex. Then the luminescence of QDs was quenched via photoinduced electron transfer by hemin associated with the particles and the electron acceptor of O2 which was in situ generated with the horseradish peroxidase-mimicked G-quadruplex/hemin DNAzymes toward H2O2. Based on this target-triggered highly efficient DNA nanoassembly and DNAzyme-modulated double quenching mechanism, the proposed biosensing strategy showed admirable signal amplification capability. Using miRNA-21 as model analyte, the designed nanosensor could detect miRNA down to 37 fM with a wide linear detection range of 1×10-13M to 1.0×10-8M, and exhibited good selectivity, acceptable reproducibility and low matrix effect. This proposed strategy presented a simple, powerful platform toward ultrasensitive miRNA detection and had great potential for bioanalysis and clinic diagnostic application.

DNA Polymerase-Directed Hairpin Assembly for Targeted Drug Delivery and Amplified Biosensing.

Due to the predictable conformation and programmable Watson-Crick base-pairing interactions, DNA has proven to be an attractive material to construct various nanostructures. Herein, we demonstrate a simple model of DNA polymerase-directed hairpin assembly (PDHA) to construct DNA nanoassemblies for versatile applications in biomedicine and biosensing. The system consists of only two hairpins, an initiator and a DNA polymerase. Upon addition of aptamer-linked initiator, the inert stems of the two hairpins are activated alternately under the direction of DNA polymerase, which thus grows into aptamer-tethered DNA nanoassemblies (AptNAs). Moreover, through incorporating fluorophores and drug-loading sites into the AptNAs, we have constructed multifunctional DNA nanoassemblies for targeted cancer therapy with high drug payloads and good biocompatibility. Interestingly, using the as-prepared AptNAs as building blocks, DNA nanohydrogels are self-assembled after centrifugation driven by liquid crystallization and dense packaging of DNA duplexes. Taking advantage of easy preparation and high loading capacity, the PDHAs are readily extended to the fabrication of a label-free biosensing platform, achieving amplified electrochemical detection of microRNA-21 (miR-21) with a detection limit as low as 0.75 fM and a dynamic range of 8 orders of magnitude. This biosensor also demonstrates excellent specificity to discriminate the target miR-21 from the control microRNAs and even the one-base mismatched one and further performs well in analyzing miR-21 in MCF-7 tumor cells.