Construction and Application of DNAzyme-based Nanodevices.

The development of stimuli-responsive nanodevices with high efficiency and specificity is very important in biosensing, drug delivery, and so on. DNAzymes are a class of DNA molecules with the specific catalytic activity. Owing to their unique catalytic activity and easy design and synthesis, the construction and application of DNAzymes-based nanodevices have attracted much attention in recent years. In this review, the classification and properties of DNAzyme are first introduced. The construction of several common kinds of DNAzyme-based nanodevices, such as DNA motors, signal amplifiers, and logic gates, is then systematically summarized. We also introduce the application of DNAzyme-based nanodevices in sensing and therapeutic fields. In addition, current limitations and future directions are discussed.

Exploring the Trans-Cleavage Activity of CRISPR/Cas12a on Gold Nanoparticles for Stable and Sensitive Biosensing.

Taking advantage of the excellent trans-cleavage activity, CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics. However, the single-stranded DNA reporter of the current CRISPR-Dx suffers from poor stability and limited sensitivity, which make their application in complex biological environments difficult. Herein, we, for the first time, explore the trans-cleavage activity of CRISPR/Cas12a toward the substrate on gold nanoparticles and apply the new phenomenon to develop a spherical nucleic acid (SNA) reporter for stable and sensitive CRISPR-Dx biosensing. By anchoring the DNA substrate on gold nanoparticles, we discovered different trans-cleavage activities of different types of the Cas12a system (e.g., LbCas12a and AsCas12a) on a nanoparticle surface. The further study suggests that the trans-cleavage activity of LbCas12a on the nanoparticle surface is highly dependent on the density and length of DNA strands. Based on these interesting discoveries, we furthermore develop SNA reporter-based fluorescent CRISPR-Dx for stable and sensitive biosensing application. Compared to traditional ssDNA reporters, the SNA reporter exhibits improved stability, which enables the stable application in a complex serum environment. In addition, the SNA reporter system with tunable density exhibits high sensitivity with a detection limit of 10 fM, which is about 2 orders of magnitude lower than that of the ssDNA reporter system. Finally, the practical application of SNA reporter-based CRISPR-Dx in clinical serum was successfully achieved. These results indicate their significant potential in future research on biology science and medical diagnoses.

Size-selective DNA nanocage-based activatable CRISPR-Cas12a for sensitive and accurate detection of mature microRNA.

The sensitive and accurate detection of mature miRNA without the signal interference by pre-miRNAs is highly important. Herein, a size-selective DNA nanocage-based activatable CRISPR/Cas12a system was developed to achieve this goal.

CRISPR-Cas12a System for Biosensing and Gene Regulation.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR-associated (Cas) proteins, Cas12a is an RNA-guided nuclease in the type V CRISPR-Cas system, which has been a robust tool for gene editing. In addition, due to the discovery of target-binding-induced indiscriminate single-stranded DNase activity of Cas12a, CRISPR-Cas12a also exhibits great promise in biosensing. This minireview not only gives a brief introduction to the mechanism of CRISPR-Cas12a but also highlights the recent developments and applications in biosensing and gene regulation. Finally, future prospects of the CRISPR-Cas12a system are also discussed. We expect this minireview will inspire innovative work on the CRISPR-Cas12a system by making full use of its features and advantages.

Size-selective molecular recognition based on a confined DNA molecular sieve using cavity-tunable framework nucleic acids.

Size selectivity is an important mechanism for molecular recognition based on the size difference between targets and non-targets. However, rational design of an artificial size-selective molecular recognition system for biological targets in living cells remains challenging. Herein, we construct a DNA molecular sieve for size-selective molecular recognition to improve the biosensing selectivity in living cells. The system consists of functional nucleic acid probes (e.g., DNAzymes, aptamers and molecular beacons) encapsulated into the inner cavity of framework nucleic acid. Thus, small target molecules are able to enter the cavity for efficient molecular recognition, while large molecules are prohibited. The system not only effectively protect probes from nuclease degradation and nonspecific proteins binding, but also successfully realize size-selective discrimination between mature microRNA and precursor microRNA in living cells. Therefore, the DNA molecular sieve provides a simple, general, efficient and controllable approach for size-selective molecular recognition in biomedical studies and clinical diagnoses.

Aptamer-Functionalized DNA Nanostructures for Biological Applications.

DNA nanostructures hold great promise for various applications due to their remarkable properties, including programmable assembly, nanometric positional precision, and dynamic structural control. The past few decades have seen the development of various kinds of DNA nanostructures that can be employed as useful tools in fields such as chemistry, materials, biology, and medicine. Aptamers are short single-stranded nucleic acids that bind to specific targets with excellent selectivity and high affinity and play critical roles in molecular recognition. Recently, many attempts have been made to integrate aptamers with DNA nanostructures for a range of biological applications. This review starts with an introduction to the features of aptamer-functionalized DNA nanostructures. The discussion then focuses on recent progress (particularly during the last five years) in the applications of these nanostructures in areas such as biosensing, bioimaging, cancer therapy, and biophysics. Finally, challenges involved in the practical application of aptamer-functionalized DNA nanostructures are discussed, and perspectives on future directions for research into and applications of aptamer-functionalized DNA nanostructures are provided.

Targeted delivery of chemically modified anti-miR-221 to hepatocellular carcinoma with negatively charged liposomes.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. Gene therapy was established as a new strategy for treating HCC. To explore the potential delivery system to support the gene therapy of HCC, negatively charged liposomal delivery system was used to deliver miR-221 antisense oligonucleotide (anti-miR-221) to the transferrin (Tf) receptor over expressed HepG2 cells. The liposome exhibited a mean particle size of 122.5 nm, zeta potential of -15.74 mV, anti-miR-221 encapsulation efficiency of 70%, and excellent colloidal stability at 4°C. Anti-miR-221-encapsulated Tf-targeted liposome demonstrated a 15-fold higher delivery efficiency compared to nontargeted liposome in HepG2 cells in vitro. Anti-miR-221 Tf-targeted liposome effectively delivered anti-miR-221 to HepG2 cells, upregulated miR-221 target genes PTEN, P27(kip1), and TIMP3, and exhibited greater silencing efficiency over nontargeted anti-miR-221 liposome. After intravenous injection into HepG2 tumor-bearing xenografted mice with Cy3-labeled anti-miR-221 Tf-targeted liposome, Cy3-anti-miR-221 was successfully delivered to the tumor site and increased the expressions of PTEN, P27(kip1), and TIMP3. Our results demonstrate that the Tf-targeted negatively charged liposome could be a potential therapeutic modality in the gene therapy of human HCC.