Receptor−ligand interactions (RLIs) that play pivotal roles in living organisms are often depicted with the classic keys-and-locks model. Nevertheless, RLIs on the cell surface are generally highly complex and nonlinear, partially due to the noncontinuous and dynamic distribution of receptors on extracellular membranes. Here, we develop a tetrahedral DNA framework (TDF)-programmed approach to topologically engineer RLIs on the cell membrane, which enables active recruitment-binding of clustered receptors for high-affinity capture of circulating tumor cells (CTCs). The four vertices of a TDF afford orthogonal anchoring of ligands with spatial organization, based on which we synthesized n-simplexes harboring 1−3 aptamers targeting epithelial cell adhesion molecule (EpCAM) that are overexpressed on the membrane of tumor cells. The 2-simplex with three aptamers not only shows increased binding affinity (∼19-fold) but prevents endocytosis by cells. By using 2-simplex as the capture probe, we demonstrate the high-efficiency CTC capture, which is challenged in real clinical breast cancer patient samples. This TDFprogrammed platform thus provides a powerful means for studying RLIs in physiological settings and for cancer diagnosis.

We have developed a surface-enhanced Raman scattering (SERS)-active substrate based on gold nanoparticle-decorated chemical vapor deposition (CVD)-growth graphene and used it for multiplexing detection of DNA. Due to the combination of gold nanoparticles and graphene, the Raman signals of dye were dramatically enhanced by this novel substrate. With the gold nanoparticles, DNA capture probes could be easily assembled on the surface of graphene films which have a drawback to directly immobilize DNA. This platform exhibits extraordinarily high sensitivity and excellent specificity for DNA detection. A detection limit as low as 10 pM is obtained. Importantly, two different DNA targets could be detected simultaneously on the same substrate just using one light source.

We present a highly sensitive colorimetric method for microRNA (miRNA) detection. This method is based on a rolling-circle amplification (RCA) DNA machine, which integrates RCA, nicking enzyme signal amplification and DNAzyme signal amplification. The DNA machine is triggered by the hybridization of target miRNA with a rational designed padlock DNA template and activated by RCA. The resulting RCA product then autonomously replicates a multiple machinery cutter cycle and generates accumulated amount of products. Specifically, the DNA product in the present work is designed as a horseradish peroxidase (HRP)-mimicking DNAzyme, which could that catalyze a colorimetric reaction and generate colored product. Through these cascade amplifications, microRNA (miRNA) as low as 2 aM could be detected. As an example of in vivo application, miRNA from single Drosophila larva was successfully analyzed. Drosophila is a model organism that provides a powerful genetic tool to study gene functions. Study of Drosophila miRNAs has brought us knowledge of its biogenesis and biological functions. The analysis of miRNA typically requires a pretreatment process of extracting total RNAs from target cells, followed by quantitative analysis of target miRNA in total RNA samples, which nevertheless suffers from laborious total RNA extraction and time-consuming processes and poor limit of detection. Meanwhile, the tiny size of Drosophila makes it difficult to accurately measure trivial changes of its cellular miRNA levels. The ability to detect ultralow concentration of miRNA of the proposed method enables the analysis the expression of mir-1 in single Drosophila larva. We thus expect that the strategy may open new avenues for in situ miRNA analysis in single cell or living animals.

Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are natural biopolymers composed of nucleotides that store, transmit, and express genetic information. Overexpressed or underexpressed as well as mutated nucleic acids have been implicated in many diseases. Therefore, nucleic acid tests (NATs) are extremely important. Inspired by intracellular DNA replication and RNA transcription, in vitro NATs have been extensively developed to improve the detection specificity, sensitivity, and simplicity. The principles of NATs can be in general classified into three categories: nucleic acid hybridization, thermal-cycle or isothermal amplification, and signal amplification. Driven by pressing needs in clinical diagnosis and prevention of infectious diseases, NATs have evolved to be a rapidly advancing field. During the past ten years, an explosive increase of research interest in both basic research and clinical translation has been witnessed. In this review, we aim to provide comprehensive coverage of the progress to analyze nucleic acids, use nucleic acids as recognition probes, construct detection devices based on nucleic acids, and utilize nucleic acids in clinical diagnosis and other important fields. We also discuss the new frontiers in the field and the challenges to be addressed.

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Weak ligand-receptor recognition events are often amplified by recruiting multiple regulatory biomolecules to the action site in biological systems. However, signal amplification in in vitro biomimetic systems generally lack the spatiotemporal regulation in vivo. Herein we report a framework nucleic acid (FNA)-programmed strategy to develop valence-controlled signal amplifiers with high modularity for ultrasensitive biosensing. We demonstrated that the FNA-programmed signal amplifiers could recruit nucleic acids, proteins, and inorganic nanoparticles in a stoichiometric manner. The valence-controlled signal amplifier enhanced the quantification ability of electrochemical biosensors, and enabled ultrasensitive detection of tumor-relevant circulating free DNA (cfDNA) with sensitivity enhancement of 3-5 orders of magnitude and improved dynamic range.

Real-time imaging of multiple low-abundance microRNAs (miRNAs) simultaneously in living cells with high sensitivity is of vital importance for accurate cancer clinical diagnosis and prognosis studies. Maintaining stability of nanoprobes resistant to enzyme degradation and enabling effective signal amplification is highly needed for in vivo imaging studies. Herein, a rationally designed one-pot assembled multicolor tetrahedral DNA frameworks (TDFs) by encoding multicomponent nucleic acid enzymes (MNAzymes) was developed for signal-amplified multiple miRNAs imaging in living cells with high sensitivity and selectivity. TDFs could enter cells via self-delivery with good biocompatibility and stability. Two kinds of MNAzymes specific for miRNA-21 and miRNA-155 with fluorescein labeling were encoded in the structure of TDFs respectively through one-step thermal annealing. In the intracellular environment, the TDFs could be specifically bound with its specific miRNA target and form an active DNAzyme structure. The cleavage of the active site would trigger the release of target miRNA and circular fluorescence signal amplification, which enabled accurate diagnosis on miRNA identifications of different cell lines with high sensitivity. Meanwhile, with the specific AS1411 aptamer targeting for nucleolin overexpressed on the surface of the carcinoma cells, this well-designed TDFs nanoprobe exhibited good discrimination between cancer cells and normal cells. The strategy provides an efficient tool for understanding the biological function of miRNAs in cancer pathogenesis and therapeutic applications.

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