Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing

Accurate measurement of intracellular temperature is of great significance in biology and medicine. With use of DNA nanotechnology and inspiration by nature’s examples of “protective and reversible responses” exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signaling mechanism, the “On” to “Off” signal is reversibly responding to “below” and “over” the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility, and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations

FRET Nanoflares for Intracellular mRNA Detection: Avoiding False Positive Signals and Minimizing Effects of System Fluctuations

A new class of intracellular nanoprobe, termed fluorescence resonance energy transfer (FRET) nanoflares, was developed to sense mRNA in living cells. It consists of a gold nanoparticle (AuNP), recognition sequences, and flares. Briefly, the AuNP functionalized with recognition sequences hybridized to flares, which are designed as hairpin structures and fluorescently labeled donors and acceptors at two ends, respectively. In the absence of targets, the flares are captured by binding with the recognition sequences, separating of the donor and acceptor, and inducing low FRET efficiency. However, in the presence of targets, the flares are gradually displaced from the recognition sequences by the targets, subsequently forming hairpin structures that bring the donor and acceptor into close proximity and result in high FRET efficiency. Compared to the conventional single-dye nanoflares, the upgraded FRET nanoflares can avoid false positive signals by chemical interferences (such as nuclease and GSH) and thermodynamic fluctuations. Moreover, the signal generation in FRET nanoflares can be easily made with ratiometric measurement, minimizing the effect of system fluctuations

Aptazyme–Gold Nanoparticle Sensor for Amplified Molecular Probing in Living Cells

To date, a few of DNAzyme-based sensors have been successfully developed in living cells; however, the intracellular aptazyme sensor has remained underdeveloped. Here, the first aptazyme sensor for amplified molecular probing in living cells is developed. A gold nanoparticle (AuNP) is modified with substrate strands hybridized to aptazyme strands. Only the target molecule can activate the aptazyme and then cleave and release the fluorophore-labeled substrate strands from the AuNP, resulting in fluorescence enhancement. The process is repeated so that each copy of target can cleave multiplex fluorophore-labeled substrate strands, amplifying the fluorescence signal. Results show that the detection limit is about 200 nM, which is 2 or 3 orders of magnitude lower than that of the reported aptamer-based adenosine triphosphate (ATP) sensors used in living cells. Furthermore, it is demonstrated that the aptazyme sensor can readily enter living cells and realize intracellular target detection

Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-Assisted Catalyzed Hairpin Assembly and “DD–A” FRET

Nucleic acids, as one kind of significant biomarker, have attracted tremendous attention and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and a donor donor–acceptor fluorescence resonance energy transfer (“DD–A” FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labeled probe as FRET donor), and H2 (TAMRA-labeled probe as FRET acceptor). First, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMB complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1–H2 duplexes were formed during the CHA process, inducing an obvious FRET signal. In contrast, CHA could not be triggered, and the FRET signal was weak, while target was absent. With the aid of magnetic separation and “DD–A” FRET, errors from background interference were effectively eliminated. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application

Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits

Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers’ attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity

Ratiometric Fluorescent Sensing of pH Values in Living Cells by Dual-Fluorophore-Labeled i‑Motif Nanoprobes

We designed a new ratiometric fluorescent nanoprobe for sensing pH values in living cells. Briefly, the nanoprobe consists of a gold nanoparticle (AuNP), short single-stranded oligonucleotides, and dual-fluorophore-labeled i-motif sequences. The short oligonucleotides are designed to bind with the i-motif sequences and immobilized on the AuNP surface via Au–S bond. At neutral pH, the dual fluorophores are separated, resulting in very low fluorescence resonance energy transfer (FRET) efficiency. At acidic pH, the i-motif strands fold into a quadruplex structure and leave the AuNP, bringing the dual fluorophores into close proximity, resulting in high FRET efficiency, which could be used as a signal for pH sensing. The nanoprobe possesses abilities of cellular transfection, enzymatic protection, fast response and quantitative pH detection. The <i>in vitro</i> and intracellular applications of the nanoprobe were demonstrated, which showed excellent response in the physiological pH range. Furthermore, our experimental results suggested that the nanoprobe showed excellent spatial and temporal resolution in living cells. We think that the ratiometric sensing strategy could potentially be applied to create a variety of new multicolor sensors for intracellular detection