Biomod/2011/LMU/FolD'N'Assemble/Results
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Summary of achievements
- The construct folded correctly.
- The cylinders dimerised correctly. The efficiency depends on the number of connections between the two cylinders
- The container openes at low pH depending on the salt concentration.
- The opening also would work in human cells because it works in PBS buffer.
- The container can be loaded by adding payload to the annealing buffer.
- Payload can be attached to the container by i-motif strands.
Folding
As a first step, we designed the construct and looked for the best folding conditions. We chose a cylindrical shape with a closed and an open end. CaDNAno-file: http://openwetware.org/wiki/Image:Nanopill.json
After some experiments, we decided to use a MgCl2 concentration of 18 mM and an annealing time of 55 h. The pictures of the TEM show that the cylinders have the right shape and size (Fig. 1).
Dimerisation
We designed four different constructs: with 2,3,4 and 5 pairs of complementary strands (Fig. 2-5). The red strands include the i-motif and the blue strands are the complementary strands.
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More connection strands led to better dimerisation (Fig. 6). With 10 connections we almost got complete dimerisation.
Pictures from the TEM show, that the dimerisation works correctly (Fig.7-8).
Opening
To open the containers we did a buffer exchange.
We exchanged the original buffer with another buffer with low pH. For good results we needed to decrease the salt concentration. With lower MgCl concentratione we got stronger monomer bands
To show that the opening process would work in the human cell, we used PBS buffer (137 mM MgCl2, which is isotonic to the human body (Fig. 10).
To check if the constructs survived the opening process we used the TEM again (Fig. 11).
Loading by statistics
After adding ssDNA+Cy5 to the annealing buffer we could show that the container is loaded (Fig. 12).
Loading by using i-motif strands
To prove, that load could be attached by our i-motif sequence an a complementary sequence, we attached ssDNA with Cy5 to the conneection strands of our construct. In Fig. 13 we can see the release of the Cy5 at low pH.
