Nucleic acids, the fundamental molecules of life, store, transmit, and express genetic information. These biomolecules are indispensable tools in biotechnology and molecular biology. The manipulation of nucleic acids, such as in vitro RNA and DNA synthesis, underpins various applications, from basic research to clinical diagnostics, gene editing, biosynthesis, drug development, and therapeutics. The workflows for these diverse applications require concentration and diafiltration (or buffer exchange) as integral steps during reaction cleanup and final formulation. These steps significantly impact the quality, yield, and downstream applications of the final product.
In the in vitro mRNA synthesis workflow, concentration and diafiltration are essential for DNA template and mRNA purification. Diluted DNA templates are concentrated, and excess salts or impurities are removed through buffer exchange to optimize conditions for subsequent reactions. Similarly, the synthesized mRNA is formulated for downstream applications such as cell transfection or storage.
In vitro mRNA synthesis and purification
Likewise, even after purification, concentration and desalting are necessary for DNA processing workflows to formulate the DNA at optimal conditions, ensuring its structural integrity and effectiveness for intended applications. Given the importance of these processes, factors like concentration and buffer exchange efficacy, gentleness, time efficiency, scalability, and final yield need to be considered when choosing appropriate methods for concentration and diafiltration of nucleic acids.
Table below presents various techniques for diafiltration and final formulation of nucleic acid, with ultrafiltration emerging as the most suitable method. However, despite being a common method, ultrafiltration using dead-end devices (dead-end filtration or DEF) is limited by low filtration rates, scalability challenges, frequent sample loss, and manual interventions.
In contrast, ultrafiltration in the form of tangential flow filtration (TFF) offers higher filtration rates and scalability. However, traditional TFF systems are typically large and have high hold-up volumes, making them unsuitable for lab scale applications. To overcome these limitations, Formulatrix has developed the µPulse® - an automated and miniaturized TFF system designed specifically for sample concentration and buffer exchange at the lab scale.
| Considerations | Precipitation |
Column Chromatography | Evaporation | Lyophilization |
Ultrafiltration Dead-End TFF |
|---|---|---|---|---|---|
| Concentration Efficacy | |||||
| Diafiltration Efficacy | |||||
| Yield | |||||
| Gentleness | |||||
| Scalability | |||||
| Processing Time | |||||
| Cost Effectiveness |
Table: Methods for nucleic acid concentration and diafiltration
Nucleic Acid Processing Using the µPulse - TFF System
The Formulatrix µPulse is an automated and miniaturized TFF system explicitly designed for lab scale applications. It is well suited for processing RNA (siRNA, mRNA), cDNA, linear, and plasmid DNA. The entire fluid path is miniaturized on the filter chip by combining our patented microfluidic pumping technology with TFF. This has reduced the hold-up volume to just 0.65 mL, ensuring maximum hold-up recovery.
The customizable settings in µPulse, including adjustable pressures and pump parameters, provide a gentle yet highly efficient nucleic acid processing. The filter chips are available with modified polyethersulfone (mPES) and regenerated cellulose (RC) membranes that exhibit low fouling characteristics and are compatible with various sample types. Furthermore, the µPulse processes samples 4x faster than the dead-end centrifugal units and in a walk-away approach.
