An Enzymatically Activated and Catalytic Hairpin Assembly-Driven Intelligent AND-Gated DNA Network for Tumor Molecular Imaging.

Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1AP, and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1AP). Subsequently, miRNA-21 facilitates the hybridization of activated H1AP on tFNA-H1AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1AP at the vertices of tFNA-H1AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.

An APE1 gated signal amplified biosensor driven by catalytic hairpin assembly for the specific imaging of microRNA in situ.

In situ imaging of microRNA (miRNA) content and distribution is valuable for monitoring tumor progression. However, tumor specific in situ imaging remains a challenge due to low miRNA abundance, lack of biological compatibility, and poor specificity. In this study, we designed a DNA tetrahedral framework complex with hairpins (DTF-HPAP) consisting of an apurinic/apyrimidinic site (AP site) that could be specifically recognized and cleaved by apurinic/apyrimidinic endonuclease 1 (APE1). Efficient and specific in situ imaging of miR-21 in tumors was thus achieved through catalytic hairpin assembly (CHA) reaction. In this study, DTF-HPAP was successfully constructed to trigger the cumulative amplification of fluorescence signal in situ. The specificity, sensitivity and serum stability of DTF-HPAP were verified in vitro, and DTF-HPAP could be easily taken up by cells, acting as a biosensor to detect tumors in mice. Furthermore, we verified the ability of DTF-HPAP to specifically image miR-21 in tumors, and demonstrated its capability for tumor-specific imaging in clinical samples.

CRISPR-Based Approaches for Efficient and Accurate Detection of SARS-CoV-2.

An outbreak of COVID-19, caused by infection with SARS-CoV-2 in Wuhan, China in December 2019, spread throughout the country and around the world, quickly. The primary detection technique for SARS-CoV-2, the reverse-transcription polymerase chain reaction (RT-PCR)-based approach, requires expensive reagents and equipment and skilled personnel. In addition, for SARS-CoV-2 detection, specimens are usually shipped to a designated laboratory for testing, which may extend the diagnosis and treatment time of patients with COVID-19. The latest research shows that clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches can quickly provide visual, rapid, ultrasensitive, and specific detection of SARS-CoV-2 at isothermal conditions. Therefore, CRISPR-based approaches are expected to be developed as attractive alternatives to conventional RT-PCR methods for the efficient and accurate detection of SARS-CoV-2. Recent advances in the field of CRISPR-based biosensing technologies for SARS-CoV-2 detection and insights into their potential use in many applications are reviewed in this article.

All-in-one approaches for rapid and highly specific quantifcation of single nucleotide polymorphisms based on ligase detection reaction using molecular beacons as turn-on probes.

Rapid, simple, specific and sensitive approaches for single nucleotide polymorphisms (SNPs) detection are essential for clinical diagnosis. In this study, all-in-one approaches, consisting of the whole detection process including ligase detection reaction (LDR) and real time quantitative polymerase chain reaction performed in one PCR tube by a one-step operation on a real-time PCR system using molecular beacon (MB) as turn-on probe, were developed for rapid, simple, specific and sensitive quantifcation of SNPs. High specificity of the all-in-one approach was achieved by using the LDR, which employs a thermostable and single-base discerning Hifi Taq DNA ligase to ligate adjacently hybridized LDR-specific probes. In addition, a highly specific probe, MB, was used to detect the products of all-in-one approach, which doubly enhances the specificity of the all-in-one approach. The linear dynamic range and high sensitivity of mutant DNA (MutDNA) and wild-type DNA (WtDNA) all-in-one approaches for the detection of MutDNA and WtDNA were studied in vitro, with a broad linear dynamic range of 0.1 fM to 1 pM and detection limits of 65.3 aM and 31.2 aM, respectively. In addition, the MutDNA and WtDNA all-in-one approaches were able to accurately detect allele frequency changes as low as 0.1%. In particular, the epidermal growth factor receptor T790M MutDNA frequency in the tissue of five patients with non-small cell lung cancer detected by all-in-one approaches were in agreement with clinical detection results, indicating the excellent practicability of the developed approaches for the quantification of SNPs in real samples. In summary, the developed all-in-one approaches exhibited promising potential for further applications in clinical diagnosis.

Pharmacophore modeling and virtual screening for the discovery of new type 4 cAMP phosphodiesterase (PDE4) inhibitors.

Type 4 cAMP phosphodiesterase (PDE4) inhibitors show a broad spectrum of anti-inflammatory effects in almost all kinds of inflamed cells, by an increase in cAMP levels which is a pivotal second messenger responsible for various biological processes. These inhibitors are now considered as the potential drugs for treatment of chronic inflammatory diseases. However, some recently marketed inhibitors e.g., roflumilast, have shown adverse effects such as nausea and emesis, thus restricting its use. In order to identify novel PDE4 inhibitors with improved therapeutic indexes, a highly correlating (r = 0.963930) pharmacophore model (Hypo1) was established on the basis of known PDE4 inhibitors. Validated Hypo1 was used in database screening to identify chemical with required pharmacophoric features. These compounds are further screened by using the rule of five, ADMET and molecular docking. Finally, twelve hits which showed good results with respect to following properties such as estimated activity, calculated drug-like properties and scores were proposed as potential leads to inhibit the PDE4 activity. Therefore, this study will not only assist in the development of new potent hits for PDE4 inhibitors, but also give a better understanding of their interaction with PDE4. On a wider scope, this will be helpful for the rational design of novel potent enzyme inhibitors.