Polyacrylamide gel electrophoresis (PAGE) is commonly used for the separation of DNA strands and complexes. For DNA nanotechnology, it is a critical characterization tool for small DNA structures1, but less useful for large DNA origami structures. PAGE gels have smaller pore sizes than agarose gel electrophoresis which gives them higher resolution for separating DNA complexes of small molecular-weight changes. However for large DNA complexes >1000 nt, the pores are too small for the DNA to enter and migrate, making separation impossible.
There are two main classes of PAGE gels: native PAGE and denaturing PAGE. Native PAGE is used for separating DNA strands or complexes of different sizes, shapes, or charges. They can be used to determine successful assembly and yield of small multi-strand complexes like DNA tetrahedrons1 or 6-strand nanopores2. They can also determine hybridization of two strands together or be used to monitor effectiveness of strand displacement reactions. Another critical application of PAGE is determining whether DNA was successfully attached to a functional unit like a protein.
Denaturing PAGE is PAGE run in the presence of urea. This is rarely used in DNA nanotechnology as the urea can disrupt the hydrogen bonding between base pairs. One use case is for ensuring staples are the expected length and no degradation or truncation products exist. The urea is useful in this case to disrupt any secondary structure that could affect the migration.
An important consideration of both AGE and PAGE is to ensure the running buffer is compatible with your structure. If the nanostructure is unstable in the running buffer, it is liable to degrade during the gel run and provide misleading results. For this reason, Mg2+ is commonly added to the running buffer.
References
- Patel, A., Valsangkar, V., Halvorsen, K. & Chandrasekaran, A. R. Purification of self-assembled DNA tetrahedra using gel electrophoresis. Curr Protoc 2, e560 (2022).
- Lanphere, C. et al. Design, assembly, and characterization of membrane-spanning DNA nanopores. Nat Protoc 16, 86–130 (2021).