Biomod/2011/TUM/TNT/LabbookA/Structure page

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Contents

Summary of all Structure related Experiments

23th Aug Folding first structures: theU BM1 and BM2
24th Aug Purification of theU structures BM1 and BM2 from 15_65 thermal ramp by agarose gel electrophoresis
29th Aug Purification of theU structures BM1 and BM2 folded with 2D_H3_ML and 5D_H3_ML via agarose gel electrophoresis
2th Sep Folding fluorophore labeled theU structures
6th Sep Size exclusion filter purification of BM_2_15_65 and BM_2_5D_H3_ML & Size exclusion filter purification of folded BM_1_15_65
12th Sep Size exclusion filter purification
27th Sep Folding fluorophore labeled structures with adapter staples for immobilisation
29th Sep purified origami structures from 27th September
17th Oct folded twisted control structure BM24
19th Oct determined concentrations of labeled oligonucleotides & folded structure BM25_4/29

List of all Origami Structures

The TUM Biomod Team 2011 folded several structures, that are described on this page.

MH_255 and MH_256

We designed an 18bp DNA double helix for reference measurements concerning the properties of the FRET labels and effects of DNA binding small molecules. The helix consists of the complementary sequences MH 255 and MH 256.

MH 255: ACGTA GACCG TTAGT ACC
MH 256: GGTAC TAACG GTCTA CGT

AT-content: 50%
TM: 53.7°C (according to supplier MWG Eurofins)

BM1 & BM2: Unlabeled Structures

With these structures, folding conditions were tested. Both structures folded properly using the most simple thermal folding ramp, 15_65.

BM1
This the_U structure contains polyT staples to prevent aggregation via stacking interactions.

BM2
The same like BM1, but without polyT staples. Observation at the transmission electron microscope displayed no negative effects of the missing polyT staples. This structure was used for the vast majority of the TEM analyses.


BM3 to BM5: Fluorophore-labeled Structures for FRET Measurements

These structures are all based on BM2. FRET labels for measurement of twists are included. The name affixes indicate the numbers of the helices carrying the labels.

BM3_4/29
This structure is fluorophore labeled for FRET measurements. Atto 550 ddCTP is attached to staple the_U 175 in helix 4 and Atto 647N ddUTP to the_U 225 in helix 29. No polyT staples are included.

BM4_8/23
This structure is fluorophore labeled for FRET measurements. Atto 550 ddCTP is attached to staple the_U 197 in helix 8 and Atto 647N ddUTP to the_U 218 in helix 23. No polyT staples are included.

BM5_5/20
This structure is fluorophore labeled for FRET measurements. Atto 550 ddCTP is attached to staple the_U 183 in helix 5 and Atto 647N ddUTP to the_U 204 in helix 20. No polyT staples are included.

BM6 to BM11: Donor-only and acceptor-only controls for the structures BM3 to BM5

BM6_4
This is the donor only control to BM3_4/29, including only Atto 550 ddCTP at the_U 175 in helix 4.

BM7_29
This is the acceptor only control to BM3_4/29, including only Atto 647N ddUTP at the_U 225 in helix 29.

BM8_8
This is the donor only control to BM4_8/23, including only Atto 550 ddCTP at the_U 197 in helix 8.

BM9_23
This is the acceptor only control to BM4_8/23, including only Atto 647N ddUTP at the_U 218 in helix 23.

BM10_5
This is the donor only control to BM5_5/20, including only Atto 550 ddCTP at the_U 183 in helix 5.

BM11_20
This is the acceptor only control to BM5_5/20, including only Atto 647N ddUTP at the_U 204 in helix 20.

BM12 to BM14: Fluorophore-labeled Structures for FRET Measurements, including Adapters for Immobilisation

The following structures all include seven special staples, which are used as adapters. With one side, they bind at he bottom of the_U, with the other side they bind to biotinylated staples, so they can be immobilised for single molecule fluorescence microscopy. The positions of the FRET labels are identical to those of structures BM3 to BM5. The name affixes indicate the numbers of the helices carrying the labels.

BM12_4/29
This structure is fluorophore labeled for FRET measurements. Atto 550 ddCTP is attached to staple the_U 175 in helix 4 and Atto 647N ddUTP to the_U 225 in helix 29. No polyT staples are included, but the seven adapter staples for immbolisiation.

BM13_8/23
This structure is fluorophore labeled for FRET measurements. Atto 550 ddCTP is attached to staple the_U 197 in helix 8 and Atto 647N ddUTP to the_U 218 in helix 23. No polyT staples are included, but the seven adapter staples for immbolisiation.

BM14_5/20
This structure is fluorophore labeled for FRET measurements. Atto 550 ddCTP is attached to staple the_U 183 in helix 5 and Atto 647N ddUTP to the_U 204 in helix 20. No polyT staples are included, but the seven adapter staples for immbolisiation.

BM15 to BM20: Donor-only and Acceptor-only Controls for the Structures BM12 to BM14

BM15_4
This is the donor only control to BM12_4/29, including only Atto 550 ddCTP at the_U 175 in helix 4.

BM16_29
This is the acceptor only control to BM12_4/29, including only Atto 647N ddUTP at the_U 225 in helix 29.

BM17_8
This is the donor only control to BM13_8/23, including only Atto 550 ddCTP at the_U 197 in helix 8.

BM18_23
This is the acceptor only control to BM17_8/23, including only Atto 647N ddUTP at the_U 218 in helix 23.

BM19_5
This is the donor only control to BM14_5/20, including only Atto 550 ddCTP at the_U 183 in helix 5.

BM20_20
This is the acceptor only control to BM14_5/20, including only Atto 647N ddUTP at the_U 204 in helix 20.

BM21 to BM23: Fluorescence labeled Structures for Length Measurement

The following structures also include the adapters for immobilisation and additionally labeled oligonucleotides to measure changes in length. The name affixes indicate the numbers of the labeled oligonucleotides.

BM21_172/173
Atto 550 ddCTP is attached to staple the_U 172 and Atto 647N ddUTP to the_U 173, both in helix 4. No polyT staples are included, but the seven adapter staples for immbolisiation.

BM22_172
This is the donor only control to BM21_172/173, including only Atto 550 ddCTP at the_U 172 in helix 4.

BM23_173
This is the acceptor only control to BM21_172/173, including only Atto 647N ddUTP at the_U 173 in helix 4.

BM24 & BM25: Twisted Structures

These structures are intrinsically twisted.

BM24
This is the twisted counterpart of BM2. No FRET labels are included. The structure was analyzed using the TEM

BM25_4/29

This is the twisted counterpart of BM12 with Atto 550 ddUTP at staple 110 in helix 4 and Atto 647N ddUTP at staple 164 in helix 29.