Research Terms
This CRISPR-Cas12a-based assay allows for the rapid, sensitive detection of microRNAs using a one-pot, isothermal process. MicroRNAs(miRNAs) are endogenous and short, non-coding single-stranded RNAs. They participate in various biological functions such as cell proliferation, differentiation, and cell death. The dysregulated expression of miRNA links to the development of different diseases, like cancer. miRNAs are found in bodily fluids, making the detection of circulating miRNAs a promising and effective strategy in liquid biopsy for cancer diagnosis, prognosis, and monitoring. However, identifying miRNA sequences is complicated by their short length and the high likelihood they closely resemble sequences from other miRNA families. While many RNA detection protocols exist, they suffer from limited practicality due to complicated workflows, the need for specialized equipment, low sensitivity, and long turnaround times.
Researchers at the University of Florida have developed EXTRA-CRISPR, a one-pot, isothermal assay for sensitively detecting miRNAs. This assay combines off-the-shelf reagents into a single reaction for fast, sensitive, and specific detection of miRNAs while reducing the chance of contamination and eliminating the need for specialized equipment.
Detection of miRNA biomarkers for point-of-care cancer diagnosis and prognosis
The core of EXTRA-CRISPR is a miRNA-specific padlock probe that detects weakly concentrated miRNA by rolling circle amplification (RCA). This padlock probe consists of single-stranded DNA with a part of its sequence complementary to the target RNA for specific identification. The probe forms a circular template around which a polymerase extends the miRNA continuously to copy the template sequence repetitively, creating a long DNA strand. Throughout these steps, EXTRA-CRISPR deploys various enzymes, including ligase, polymerase, and Cas12a, to amplify the amount of miRNA, facilitating the detection of miRNA biomarkers.
Ligase transforms the padlock probe into a circular template, with miRNA serving as a splint. Polymerase enables RCA, and the cis-cleavage activity of Cas12a cuts the RCA product, generating cleaved segments that exponentially trigger secondary amplification.
At the same time, Cas12a can cleave a fluorescent reporter using its trans-cleavage activity, generating a fluorescent signal detectable in real-time by standard quantitative polymerase chain reaction tools or even with smartphone applications. This streamlined process of miRNA detection enhances the feasibility of using miRNAs for point-of-care diagnostics.
This method for modifying extracellular vesicles combines enzymatic glycan engineering with chemoselective functionalization to unlock applications such as targeted drug delivery. Extracellular vesicles are lipid bilayer-enclosed nanoparticles secreted by cells, acting as important messengers and substance transporters between cells. However, they must be modified to carry special molecular markers and functional cargos for practical application. Although many different modification methods exist, such as introducing lipophilic molecules to the vesicle’s membrane, they are difficult to control and often inadvertently influence the membrane structure or other properties of extracellular vesicles. Glycans or carbohydrates are prevalent and exposed on the surface of extracellular vesicles, rendering them ideal facilitators for modifying extracellular vesicles.
Researchers at the University of Florida have developed a method using enzymatic reactions to modify the surface glycans of extracellular vesicles. By introducing functionalized sugar residues, they can label carbohydrates on the surface of extracellular vesicles and enable the attachment of various molecules, including affinity and fluorescent tags, ligands, antigens, antibodies, and drugs.
Functionalizing extracellular vesicles for biomedical applications such as immunotherapy and targeted drug delivery
The basic objective of extracellular vesicle engineering is attaching functional groups to the surface of the extracellular vesicles. These functional groups serve diverse purposes such as labeling vesicles with specific tags, ligands, and antibodies for targeted drug delivery or cancer immunotherapy. The glycans coating extracellular vesicles provides a straightforward method to functionalization via enzymatic glycan engineering. The glycans serve as the attachment point for functionalization owing to their abundance and excellent locations on the extracellular vesicle surface and their flexibility as the first link in the functional chain. Accordingly, an unnatural sugar residue with an azide is enzymatically attached to the natural sugar residue, facilitating further modification with various molecules for biological applications. The enzyme that enters this functionalization is a glycosyltransferase, which catalyzes the formation of covalent bonds between sugars, called glycosidic bonds. This enzyme proves especially wide-reaching in its ability to introduce useful unnatural sugar residues into the glycan chains.
