Isolation of DNA aptamers using micro free flow electrophoresis.

As we have seen in Gavin's class, Systematic Evolution of Ligands by EXponential enrichment, or SELEX, could be used to find the aptamer portion of a riboswitch. This is the traditional way to find RNA or DNA sequences that bind to small molecules or proteins. However, there can be problems with this selection which include: having to bind the small molecule to a solid support and high dilution ratios.
In this paper, the authors use a new method called micro free flow electrophoresis to find aptamers against IgE. This device works by separating bound aptamers from unbound aptmers by using an electrical charge, where bound aptamers should be less anionic and migrate differently. The figure below shows the best binds are found after the first round of selection without the need to do a negative selection. The authors also found the dilution to be only 100 fold which is 60 fold less than what is seen in a SELEX experiment.

Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification

This is the paper that we discussed in class regarding the PylRS crystal structure and orthogonality. They state "The M. mazei PylRS and tRNA^Pyl are orthogonal not only to the bacterial systems but also to the eukaryotic systems, and thus these lysine derivatives have been successfully incorporated into a protein in yeast and mammalian cells" The way it is written makes it sound as though the other PylRS/tRNA pairs are not orthogonal in mammalian cells. All of the lysine analogue incorporation that involved mammalian cells i reviewed were using the M. mazei PylRS.