I am performing cell free protein synthesis of GFP with commercial RTS 5-Prime kit using DNA-modified hyaluronic acid-PEG hydrogel particles. The fluorescently labeled DNA is covalently coupled to the gel and can be clearly detected with confocal microscopy. However, when I perform gene expression on the plate reader I do not see any increase of fluorescence intensity. As a control I perform CFPS with the same DNA template in solution and this works out perfectly. Why is there no gene expression?
It is possible that cross-linking the DNA to a solid support may prevent unwinding or present some other structural impediment to transcription. I would assume the cross links are at the ends, 5' or 3'. If so, are both strands linked? Might be best to only cross link one of the strands.
Gregory Dressler
The 5' is functionalized with SH to couple covalently to the gel, 3' is modified with Atto565 dye to visualize the DNA on the gel.
Perhaps there is an unspecific adsorption on the solid support? DNA/proteins may also interact too tightly with the surface (ionic/counter ionic layers). Is there a long enough DNA linker?
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