Programming a DNA tetrahedral nanomachine as an integrative tool for intracellular microRNA biosensing and stimulus-unlocked target regulation.

The synchronous detection and regulation of microRNAs (miRNAs) are essential for the early tumor diagnosis and treatment but remains a challenge. An integrative DNA tetrahedral nanomachine was self-assembled for sensitive detection and negative feedback regulation of intracellular miRNAs. This nanomachine comprised a DNA tetrahedron nanostructure as the framework, and a miRNA inhibitor-controlled allosteric DNAzyme as the core. The DNA tetrahedron brought the DNAzyme and the substrate in spatial proximity and facilitated the cellular uptake of DNAzyme. In allosteric regulation of DNAzyme, the locked tetrahedral DNAzyme (L-tetra-D) and active tetrahedral DNAzyme (A-Tetra-D) were controlled by miRNA inhibitor. The combination of miRNA inhibitor and target could trigger the conformational change from L-tetra-D to A-Tetra-D. A-Tetra-D cleaved the substrate and released fluorescence for intracellular miRNA biosensing. The DNA tetrahedral nanomachine showed excellent sensitivity (with detection limit down to 0.77 pM), specificity (with one-base mismatch discrimination), biocompatibility and stability. Simultaneously, miRNA stimulus-unlocked inhibitor introduced by our nanomachine exhibited the synchronous regulation of target cells, of which regulatory performance has been verified by the upregulated levels of downstream genes/proteins and the increased cellular apoptosis. Our study demonstrated that the DNA tetrahedral nanomachine is a promising biosense-and-treat tool for the synchronous detection and regulation of intracellular miRNA, and is expected to be applied in the early diagnosis and tailored management of cancers.

Enzyme-free and copper-free strategy based on cyclic click chemical-triggered hairpin stacking circuit for accurate detection of circulating microRNAs.

Accurate detection of circulating microRNAs (miRNAs) plays a vital role in the diagnosis of various diseases. However, enzyme-free amplification detection remains challenging. Here, we report an enzyme-free fluorescence resonance energy transfer assay termed "3C-TASK" (cyclic click chemical-triggered hairpin stacking kit) for the detection of circulating miRNA. In this strategy, the miRNA could initiate copper-free click chemical ligation reactions and the ligated products then trigger another hairpin stacking circuit. The first signal amplification was achieved through the recycling of the target miRNA in the click chemical ligation circuit, and the second signal amplification was realized through the recycling of ligated probes in a hairpin stacking circuit driven by thermodynamics. The two-step chain reaction event triggered by miRNAs was quantified by the fluorescence signal value so that accurate detection of target miRNA could be achieved. The 3C-TASK was easily controlled because no enzyme was involved in the entire procedure. Although simple, this strategy showed sensitivity with a detection limit of 8.63 pM and specificity for distinguishing miRNA sequences with single-base variations. In addition, the applicability of this method in complex biological samples was verified by detecting target miRNA in diluted plasma samples. Hence, our method achieved sensitive and specific detection of miRNA and may offer a new perspective for the broader application of enzyme-free chemical reaction and DNA circuits in biosensing.

A multiplexed circulating tumor DNA detection platform engineered from 3D-coded interlocked DNA rings.

Circulating tumor DNA (ctDNA) is a critical biomarker not only important for the early detection of tumors but also invaluable for personalized treatments. Currently ctDNA detection relies on sequencing. Here, a platform termed three-dimensional-coded interlocked DNA rings (3D-coded ID rings) was created for multiplexed ctDNA identification. The ID rings provide a ctDNA recognition ring that is physically interlocked with a reporter ring. The specific binding of ctDNA to the recognition ring initiates target-responsive cutting via a restriction endonuclease; the cutting then triggers rolling circle amplification on the reporter ring. The signals are further integrated with internal 3D codes for multiplexed readouts. ctDNAs from non-invasive clinical specimens including plasma, feces, and urine were detected and validated at a sensitivity much higher than those obtained through sequencing. This 3D-coded ID ring platform can detect any multiple DNA fragments simultaneously without sequencing. We envision that our platform will facilitate the implementation of future personalized/precision medicine.

Boolean logic gate based on DNA strand displacement for biosensing: current and emerging strategies.

DNA computers are considered one of the most prominent next-generation molecular computers that perform Boolean logic using DNA elements. DNA-based Boolean logic gates, especially DNA strand displacement-based logic gates (SDLGs), have shown tremendous potential in biosensing since they can perform the logic analysis of multi-targets simultaneously. Moreover, SDLG biosensors generate a unique output in the form of YES/NO, which is contrary to the quantitative measurement used in common biosensors. In this review, the recent achievements of SDLG biosensing strategies are summarized. Initially, the development and mechanisms of Boolean logic gates, strand-displacement reaction, and SDLGs are introduced. Afterwards, the diversified input and output of SDLG biosensors are elaborated. Then, the state-of-the-art SDLG biosensors are reviewed in the classification of different signal-amplification methods, such as rolling circle amplification, catalytic hairpin assembly, strand-displacement amplification, DNA molecular machines, and DNAzymes. Most importantly, limitations and future trends are discussed. The technology reviewed here is a promising tool for multi-input analysis and lays a foundation for intelligent diagnostics.

3D DNA nanonet structure coupled with target-catalyzed hairpin assembly for dual-signal synergistically amplified electrochemical sensing of circulating microRNA.

DNA nanomaterials are reliable and powerful tools in the development of a variety of biosensors owing to their notable self-assembly ability and precise recognition capability. Here, we propose a DNA nanomaterial-based system for the dual-amplified electrochemical sensing of circulating microRNAs by a coupled cascade of catalyzed hairpin assembly (CHA) and three-dimensional (3D) DNA nanonet structure. In the target-assisted CHA process, the stable hairpin structures H1 and H2 act as probes for the recognition and recycling of circulating microRNAs, leading to the formation of abundant H1-H2 duplexes with tails. Subsequently, a 3D DNA nanonet structure was introduced, which was assembled using three DNA strands constructed X-DNA monomers as the building blocks, and hybridized to the tails of H1-H2 duplexes. The successful integration of target-assisted CHA and 3D DNA nanonet structure induced the second signal amplification. The designed biosensor performed under optimized experimental conditions, and exposed admirable analytical performance for the detection of circulating miR-21, with a wide linear range from 10 fM to 1 nM, high sensitivity of limit of detection (LOD) of 3.6083 fM, good specificity in the face of single nucleotides and other microRNAs, satisfactory stability and reproducibility for practical analysis. Furthermore, the clinical applicability for circulating miR-21 detection was verified in human serum samples without additional treatment. We hope that this elaborated biosensor will provide new opportunities for bioassays based on DNA nanomaterials.

Strand displacement-triggered G-quadruplex/rolling circle amplification strategy for the ultra-sensitive electrochemical sensing of exosomal microRNAs.

Emerging evidence suggests that exosomal microRNAs are potential biomarkers for the early diagnosis and prognostic assessment of tumor. Here, we design a strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy for highly specific and sensitive electrochemical sensing of exosomal microRNAs. In the presence of exosomal miRNA-21, a locked nucleic acid (LNA)-labeled toehold mediated strand displacement reaction (TMSDR) is initiated, releasing output P2 to trigger the subsequent RCA reaction by hybridizing with the C-rich circular template. Then the obtained G-rich RCA products can bind to the probe anchored on the surface of gold electrode and generate G-quadruplex conformations. Based on the TMSDR-triggered G-quadruplex/RCA strategy, the detection limit of this electrochemical biosensor is down to 2.75 fM. Moreover, our biosensor exhibits excellent repeatability, stability, and high consistency compared to RT-PCR for clinical detection. In conclusion, this assay is expected to provide a hopeful strategy for the early non-invasive diagnosis and prognostic estimation of cancer. Graphical abstract Schematic illustration of electrochemical sensing of exosomal microRNAs based on strand displacement-initiated G-quadruplex/rolling circle amplification (RCA) strategy.