MicroRNA-Responsive DNA-Programmed Nanomedicine with Controllability of Cascaded Events for Cancer Therapy Enhancement.

Chemotherapy is a prime tool for cancer clinical therapy. The effectiveness has been improved considerably with the assistance of nanotechnology. However, it still meets the challenge of unsatisfied therapeutic effects caused by multidrug resistance and uncontrollable drug release. For further enhancement of the treatment performance, we develop a kind of microRNA-responsive nanomedicine that uses the biomarker microRNA-21 as a trigger of cascaded killing effects on cancer cells, including chemotherapy and gene silencing. The nanomedicine consists of a gold nanoparticle core and a DNA layer. Strand migrations within the layer can accurately control the events of anticancer drug doxorubicin release and multidrug-resistant-associated protein 1 downregulation, yielding an alleviation of multidrug resistance and enhanced killing on cancer cells. This work demonstrates a microRNA-responsive nanomedicine in combination with chemotherapy and gene silencing, which paves the way to the advancement of DNA-based nanomedicine for cancer theranostics.

A novel analytical principle using AP site-mediated T7 RNA polymerase transcription regulation for sensing uracil-DNA glycosylase activity.

Uracil DNA glycosylase (UDG) is a highly conserved damage repair glycosylase; the abnormal expression of DNA glycosylase has important research value in many human diseases. Therefore, highly sensitive and specific detection of UDG activity is crucial to biomedical research and clinical diagnosis. In this work, we propose an AP site-mediated T7 RNA polymerase transcription regulation analytical principle for uracil-DNA glycosylase activity analysis. T7 RNA polymerase is highly promoter-specific and only transcribes DNA downstream of the T7 promoter. We have found that modifying the T7 promoter sequence with an AP site can regulate T7 RNA polymerase transcription ability according to different modification sites. In the binding region of the promoter, AP sites greatly inhibit transcription. Moreover, AP sites in the initiation region of the promoter enhance transcription activity. Based on this research, we designed a new transcription substrate template by replacing deoxythymidine (dT) in the T7 RNA polymerase promoter sequence with one tetrahydrofuran abasic site mimic (THF) and one deoxyuridine (dU). The THF site was labeled in the transcription-enhanced region to improve transcription background, and the dU site was labeled in the transcription inhibition region to sense the UDG enzyme. In our strategy, this template can be transcribed into RNAs by T7 RNA polymerase with great multicycle amplifications. When UDG is present, dU is excised to form an AP site. The AP site damages the interaction between T7 RNA polymerase and the T7 promoter, resulting in weak transcription activity. The detection limit of this strategy is as low as 2.5 × 10-4 U mL-1, and it has good selectivity for UDG. In addition, this strategy can also detect UDG activity in complex HeLa cell lysate samples. Therefore, our developed sensor might become a promising technique for UDG activity assay.

A fluorescent signal "removal" sensor via duplex-specific nuclease-aided cleavage for miRNA detection in flow cytometry.

Considered as the next-generation biomarkers, microRNAs play an important role in the early diagnosis of cancers. Here, we designed a fluorescent signal "removal" sensor for one-step, sensitive and specific detection of multiple microRNAs by flow cytometry (FCM). In this work, single-stranded DNA (ssDNA), working as the interlinkage, immobilized the fluorescent nanosphere (FS) onto the SiO2 microspheres surface to form the SiO2-ssDNA-FS probes. When target miRNAs integrated with SiO2-ssDNA-FS probes, the duplex-specific nuclease (DSN) could cleave the ssDNA selectively and release FS with numerous cycles to enhance the fluorescent signal attenuation of SiO2-ssDNA-FS, so as to remarkably improve the analysis sensitivity. It achieved a simple, accurate and quantitative microRNA-21 detection for clinical blood samples. Parallel multi-target detection of microRNA-21 and Let-7d was also realized by different color labeled FS. Moreover, our designed sensor was suitable for other targets' detection with the corresponding probes.

Reusable Bioluminescent Sensor for Ultrasensitive MicroRNA Detection Based on a Target-Introducing "Fuel-Loading" Mechanism.

As a kind of important potential biomarkers, the expression level of some microRNAs (miRNAs) is closely related to cancer development and progression. Herein, a reusable ultra-sensitive "fuel-loadings" bioluminescent sensor was constructed to detect the trace miRNA based on the cascading signal amplification, which combined the target-introducing "fuel-loading" mechanism and cyclic bioluminescence assay. In this sensor, magnetic beads labeled with hairpin DNA probes (hDNA) could specifically hybridize with the target miRNA and isolate these targets from samples. Then, the target-introducing "fuel loading" mechanism worked because the poly(A) polymerase can catalyze the template-independent sequential addition of adenosine monophosphate (AMP) to the 3' ends of the miRNA targets to produce long poly(A) tails. The long poly(A) tails provided lots of 5'AMPs (cleaved by Exonuclease T), which further as fuels were converted into adenosine-triphosphate (ATP) to generate an enhanced bioluminescent signal by cyclic AMP pyrophosphorylation-ATP dephosphorylation. The "fuel-loadings" bioluminescent sensor realized a high sensitivity with a limit-of-detection of about 22.6 aM for miRNA 21. Moreover, this "fuel-loadings" bioluminescent sensor not only achieved regenerable and reusable measurement in the same microwell to decrease the analysis costs, but also could directly detect miRNA 21 in the serum without complicated extraction procedures. It showed excellent coherence with quantitative reverse transcription polymerase chain reaction for miRNA 21 detection of cancer patients' samples, indicating clinical translation potential for miRNA detection.