Biomod/2011/LMU/FolD'N'Assemble/Results

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HOME
THE PROJECT
METHODS
PROTOCOLS
LAB BOOK
RESULTS
TEAM

Timon Funck

Aleksej Belizki

Ralf Weidner

Miranda Roßmann

Verena Schüller

Prof. Tim Liedl


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).


Fig.1: The monomers have the designed size and shape
Fig.1: The monomers have the designed size and shape

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.

4 connections
4 connections
6 connections
6 connections
8 connections
8 connections
10 connections
10 connections

More connection strands led to better dimerisation (Fig. 6). With 10 connections we almost got complete dimerisation.


Fig. 6: 2% agarose gel. With more connection we get better dimerisation
Fig. 6: 2% agarose gel. With more connection we get better dimerisation

Pictures from the TEM show, that the dimerisation works correctly (Fig.7-8).

Fig.7: Dimers with 10 connections
Fig.7: Dimers with 10 connections
Fig.8: Dimers with 4 connections
Fig.8: Dimers with 4 connections

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

Fig.9: 2% agarode gel. The monomer bandes become stronger for lower salt concentrations
Fig.9: 2% agarode gel. The monomer bandes become stronger for lower salt concentrations


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).


Fig.10: 2% agarose gel. The samples with low pH have almost no dimers any more. The lanes with lower pH run slower because of the positive charge of the Hydrogen ions
Fig.10: 2% agarose gel. The samples with low pH have almost no dimers any more. The lanes with lower pH run slower because of the positive charge of the Hydrogen ions

To check if the constructs survived the opening process we used the TEM again (Fig. 11).

Fig.11: Monomers after opening process
Fig.11: Monomers after opening process

Loading by statistics

After adding ssDNA+Cy5 to the annealing buffer we could show that the container is loaded (Fig. 12).

Fig. 12: Laser scanner (right) and florescence(left) image with Cy5 loaded dimers, 2% agarose gel, 3h running time
Fig. 12: Laser scanner (right) and florescence(left) image with Cy5 loaded dimers, 2% agarose gel, 3h running time

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.


Fig. 13: Laser scanner(left) and UV florescence(right) of Cy5 strands attached to the monomer, 2% agarose gel, 3h running time, left to right, monomers, monomers after buffer exchange pH 8, monomers after buffer exchange pH 4.5
Fig. 13: Laser scanner(left) and UV florescence(right) of Cy5 strands attached to the monomer, 2% agarose gel, 3h running time, left to right, monomers, monomers after buffer exchange pH 8, monomers after buffer exchange pH 4.5
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