CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

DNA-scaffolded multivalent vaccine against SARS-CoV-2.

Short peptides are poor immunogens. One way to increase their immune responses is by arraying immunogens in multivalency. Simple and efficient scaffolds for spatial controlling the inter-antigen distance and enhancing immune activation are required. Here, we report a molecular vaccine design principle that maximally drives potent SARS-CoV-2 RBD subunit vaccine on DNA duplex to induce robust and efficacious immune responses in vivo. We expect that the DNA-peptide epitope platform represents a facile and generalizable strategy to enhance the immune response. STATEMENT OF SIGNIFICANCE: DNA scaffolds offer a biocompatible and convenient platform for arraying immunogens in multivalency antigenic peptides, and spatially control the inter-antigen distance. This can effectively enhance immune response. Peptide (instead of entire protein) vaccines are highly attractive. However, short peptides are poor immunogens. Our DNA scaffolded multivalent peptide immunogen system induced robust and efficacious immune response in vivo as demonstrated by the antigenic peptide against SARS-CoV-2. The present strategy could be readily generalized and adapted to prepare multivalent vaccines against other viruses or disease. Particularly, the different antigens could be integrated into one single vaccine and lead to super-vaccines that can protect the host from multiple different viruses or multiple variants of the same virus.

2D MOF-Based Photoelectrochemical Aptasensor for SARS-CoV-2 Spike Glycoprotein Detection.

A reliable and sensitive detection approach for SARS-CoV 2 is essential for timely infection diagnosis and transmission prevention. Here, a two-dimensional (2D) metal-organic framework (MOF)-based photoelectrochemical (PEC) aptasensor with high sensitivity and stability for SARS-CoV 2 spike glycoprotein (S protein) detection was developed. The PEC aptasensor was constructed by a plasmon-enhanced photoactive material (namely, Au NPs/Yb-TCPP) with a specific DNA aptamer against S protein. The Au NPs/Yb-TCPP fabricated by in situ growth of Au NPs on the surface of 2D Yb-TCPP nanosheets showed a high electron-hole (e-h) separation efficiency due to the enhancement effect of plasmon, resulting in excellent photoelectric performance. The modified DNA aptamer on the surface of Au NPs/Yb-TCPP can bind with S protein with high selectivity, thus decreasing the photocurrent of the system due to the high steric hindrance and low conductivity of the S protein. The established PEC aptasensor demonstrated a highly sensitive detection for S protein with a linear response range of 0.5-8 µg/mL with a detection limit of 72 ng/mL. This work presented a promising way for the detection of SARS-CoV 2, which may conduce to the impetus of clinic diagnostics.