Isolation of Small Regulatory RNA–Bound Bacterial Target RNA Using Affinity Purification

Take a bacterial cell lysate containing cellular components, including MS2-tagged sRNA bound to its target RNA.

MS2-tagged sRNA is an engineered small regulatory RNA carrying an MS2 aptamer stem-loop at its 5′ end, which exhibits high affinity for the MS2 viral coat protein.

Transfer the bacterial lysate to a pre-equilibrated affinity chromatography column containing amylose resin bound to fusion proteins composed of maltose-binding protein (MBP) and the MS2 coat protein.

The immobilized MS2 coat protein specifically captures the aptamer stem-loop of the MS2-tagged sRNA, retaining the intact target RNA-bound sRNA.

Untagged molecules that remain unbound are collected in the flow-through.

Wash the column with buffer to remove residual unbound molecules.

Elute with a buffer containing maltose, which displaces MBP from the resin, releasing the MS2-tagged sRNA bound to its target RNA.

Collect and store the fractions on ice to preserve RNA integrity for downstream analysis.

Remove the column tip, then put a chromatography column in a column rack and wash the column with ultrapure water. Add 300 microliters of amylose resin and wash the column with 10 milliliters of buffer A.Dilute 1, 200 picamoles of MBP-MS2 protein in six milliliters of buffer A and load it into the column.

Wash the column with 10 milliliters of buffer A.Next, perform MS2-affinity purification. Load the cell lysate into the column and collect the flow-through fraction in a clean collection tube. Wash the column three times with 10 milliliters of buffer A and collect the wash fraction, then elute the column with one milliliter of buffer E and collect the elution fraction in a two milliliter microtube.

Keep all collected fractions on ice until RNA extraction.