(188bi) Charge-Switch Membranes for the Rapid Isolation of microRNA

Small regulatory RNAs called microRNA (miRNA) are essential components of cellular function, likely regulating >60% of mammalian protein-encoding genes. miRNAs are dysregulated in a wide variety of diseases including major depressive disorder, various cancers, neurodegenerative diseases, autoimmune disorders, cardiovascular diseases, and infectious disease. As miRNA are both ubiquitous and stable in a large range of biological fluids, they have vast potential as diagnostic biomarkers and in fundamental biological research. At approximately 22 nucleotides long, miRNA extraction poses unique challenges. Additionally, miRNAs are often present in low concentrations in biological samples, such that concentrating small RNA during extraction is advantageous. Current kits require centrifugation or magnetic bead extraction, organic solvents, toxic chaotropic salts, and 15 to 45 minutes to isolate and purify miRNA.

Positively charged polymers have shown great promise in DNA and RNA extraction, as these protocols do not require chaotropic salts or organic solvents to purify nucleic acids. Chitosan is one such polymer that is frequently used to capture and delivery DNA. The primary amines along the chitosan backbone are positively charged at low pH (pKa ~ 6.3), and are neutral at high pH. The membrane therefore is positively charged and capable of binding the negatively charged phosphate backbone of DNA and RNA at low pH, and can elute these nucleic acids at a high pH. While promising, this method has not yet been extended to miRNA. We have developed a chitosan-functionalized cellulose membrane to rapidly isolate and concentrate miRNA. Varying chitosan chain length and fabrication techniques controls the surface density of chitosan-derived amine groups on the membrane. These membranes can rapidly capture 89% of a labeled 20 nucleotide ssDNA. We are extending this method to rapidly isolate clinically relevant miRNA that are putative biomarkers for Traumatic Brain Injury.