Development of smart DNA nanostructures is of great value in cancer studies. Here, by integrating rolling circle amplification (RCA) into split aptamer design, a novel strategy of polyvalent and thermosensitive DNA nanoensembles was first proposed for cancer cell detection and manipulation. In this strategy, a long nanosolo ssDNA with repeated Split-b and Poly T regions was generated through RCA. Split-b supplied polyvalent binding sites while Poly T supported signal output by hybridizing with fluorophore-labeled poly A. After addition of Split-a, nanoensembles formed on the cell surface due to target-induced assembly of Split-a/Split-b from the free state to the recognition structure, and on the basis of the thermosensitivity of split aptamer, nanoensembles were controlled reversibly by changing temperatures. As proof of concept, split ZY11 against SMMC-7721 cancer was used to construct nanoensembles. Compared with monovalent split aptamer, nanoensembles were demonstrated to have a much stronger interaction with target cells, thus realizing an ∼2.8-time increase in signal-to-background ratio (SBR). Moreover, nanoensembles extended the tolerance range of target binding from 4 °C to room temperature and speeded recognition thus achieving almost 50% binding in 1 min. Then, nanoensembles were successfully applied to detect 7721 cells in serum and mixed cell samples. By utilizing microplate well surface as the model, temperature-controlled catch/release of target cells was also realized with nanoensembles, even under unfriendly conditions for monovalent split aptamer. The RCA-mediated aptameric nanoensembles strategy not only solved the problem of split aptamer in inefficient binding but also paved a brand new way for developing polyvalent and intelligent nanomaterials.

•A green, facile, low-cost, label-free and ultrasensitive assay for DNase I activity.•A seamless integration of DNase I digestion and TdT polymerization.•TdT-polymerized superlong poly T ssDNA (>500 mer) for fluorescent CuNPs formation.•A limit of detection as low as 0.02 U/mL was realized for DNase I analysis.•DNase I in diluted serum was successfully detected with superlong poly T-CuNPs.Deoxyribonuclease I (DNase I) is an important physiological indicator and diagnostic biomarker, but traditional methods for assessing its activity are time-consuming, laborious, and usually radioactive. Herein, by effectively combining the special functions of DNase I and terminal deoxynucleotidyl transferase (TdT), a simple, green, cost-effective, label-free and ultrasensitive assay for DNase I activity has been constructed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). In this strategy, a 3′-phosphorylated DNA primer is designed to block TdT polymerization. After addition of DNase I, the primer could be digested to release 3′-hydroxylated fragments, which could further be tailed by TdT in dTTP pool with superlong poly T ssDNA for CuNPs formation. Fluorescence measurements and gel electrophoresis demonstrated its feasibility for DNase I analysis. The results indicated that with a size of 3–4 nm, the CuNPs templated by TdT-polymerized superlong poly T (>500 mer) had several advantages such as short synthetic time (<5 min), large Stokes shift (~275 nm) and intense red fluorescence emission. Under the optimal conditions, quantitative detection of DNase I was realized, showing a good linear correlation between 0.02 and 2.0 U/mL (R2=0.9928) and a detection limit of 0.02 U/mL. By selecting six other nucleases or proteins as controls, an excellent specificity was also verified. Then, the strategy was successfully applied to detect DNase I in diluted serum with a standard addition method, thus implying its reliability and practicability for biological samples. The proposed strategy might be promising as a sensing platform for related molecular biology and disease studies.By seamlessly integrating the special functions of deoxyribonuclease I (DNase I) and terminal deoxynucleotidyl transferase (TdT), a novel fluorescent sensor for DNase I activity has been developed based on superlong poly(thymine)-hosted copper nanoparticles (poly T-CuNPs). The strategy is green, facile, cost-effective, label-free and highly-sensitive, thus achieving a limit of detection as low as 0.02 U/mL and potential applicability to biological samples.Download high-res image (156KB)Download full-size image

A label-free and general thermo-controlled split apta-PCR strategy was first developed for the sensitive and specific detection of cancer cells. By integrating the temperature-responsive function of split aptamers with PCR amplification, a facile fluorescence assay of liver cancer SMMC-7721 cells was successfully realized with the detection of as low as 100 cells.

DNA-based activatable theranostic nanoprobes are still unmet for in vivo applications. Here, by utilizing the “induced-fit effect”, a smart split aptamer-based activatable theranostic probe (SATP) was first designed as “nanodoctor” for cancer-activated in vivo imaging and in situ drug release. The SATP assembled with quenched fluorescence and stable drug loading in its free state. Once binding to target proteins on cell surface, the SATP disassembled due to recognition-triggered reassembly of split aptamers with activated signals and freed drugs. As proof of concept, split Sgc8c against CEM cancer was used for theranostic studies. Benefiting from the design without blocking aptamer sequence, the SATP maintained an excellent recognition ability similar to intact Sgc8c. An “incubate-and-detect” assay showed that the SATP could significantly lower background and improve signal-to-background ratio (∼4.8 times of “always on” probes), thus affording high sensitivity for CEM cell analysis with 46 cells detected. Also, its high selectivity to target cells was demonstrated in analyzing mixed cell samples and serum samples. Then, using doxorubicin as a model, highly specific drug delivery and cell killing was realized with minimized toxicity to nontarget cells. Moreover, in vivo and ex vivo investigations also revealed that the SATP was specifically activated by CEM tumors inside mice. Especially, contrast-enhanced imaging was achieved in as short as 5 min, thus, laying a foundation for rapid diagnosis and timely therapy. As a biocompatible and target-activatable strategy, the SATP may be widely applied in cancer theranostics.