A critical challenge faced during the development and manufacturing of RNA-based therapies is the presence of unintended impurities, particularly double-stranded RNA (dsRNA). While dsRNA is an inevitable byproduct of the in vitro transcription (IVT) process, unchecked contamination can severely jeopardize both the safety and effectiveness of the treatment
This protocol describes a method for coupling ligands containing hydrazine, hydrazide, aminooxy, or amine groups to aldehyde-containing particles. Aldehyde particles are reactive with hydrazine, hydrazide, or aminooxy derivatives, forming hydrazone or oxime linkages. Reactions with amine-containing molecules, such as proteins, can be carried out through a reductive amination process using sodium cyanoborohydride.
This protocol describes a method for coupling ligands containing thiol, amino, or hydroxyl groups to epoxide-containing particles via a ring-opening reaction that is facilitated under alkaline conditions. This reactive group can be used to couple proteins, nucleic acids, sugars and carbohydrates, and other organic molecules containing these functionalities.
This protocol describes methods for covalently binding ligands to hydroxyl particles. Since hydroxyl groups are not spontaneously reactive with biomolecules, they must be activated using a variety of methods. Most activation strategies are carried out under nonaqueous conditions because the activating agent and the reactive intermediate group are typically susceptible to hydrolysis.
An alternative method for coupling thiol-containing proteins or antibodies to amine particles is to use a heterobifunctional crosslinker containing an amine-reactive NHS ester at one end and a thiol-reactive maleimide group on the other end. NHS–PEGn–maleimide crosslinkers are available in a number of spacer lengths depending on the size of the polymer chain in the PEG component
The crosslinker SPDP can be reacted with amine particles to create thiol-reactive pyridyl disulfide groups on the surface. Thiol-containing proteins or other thiol molecules can be reacted with these activated particles to result in disulfide linkages, which are reversible by reduction
Glutaraldehyde-mediated immobilization onto amine particles can be performed with some control using two main reaction routes.
This protocol outlines a general method for covalently conjugating biomolecules to amine-modified beads. It uses a homobifunctional crosslinker, such as glutaraldehyde, imidoesters, or NHS esters, to link available amine groups on a ligand to the amine-functionalized beads.
