First Click Reactions

LABBOOK

Optimization of the folding yield

We decided to put our original labbook online to promote scientific integrity. Furthermore we have no intention to hide negative results, so this page shows our entire, unmodified work.

Bio 13 (July 2nd)

Materials:

Bio 14 (July 3rd)

Bio 15 (July 9th)

Bio 16 (July 10th)

All Chemicals were supplied by Aldrich, Fluka or Roth if not stated otherwise. The alkyne DNA staples were supplied by BaseClick as well as the OligoClick kit. The 2-Azidoethyl alpha-Mannopyranoside was supplied by Sussex Research. Other azide sugars were supplied by Glycan Array Synthesis Core (D). The Aminoallyl-dUTP - Cy5, Aminoallyl-dUTP - ATTO-550 and Aminoallyl-dUTP-XX - ATTO-488 was supplied by Jena Bioscience. The Reversed Transcriptase was supplied by Roche. Short oligonucleotides where supplied by MWG Eurofins.

Bio 25 (August 5th)

Bio 26 (August 6th)

Bio 27 (August 11th)

First Click Reactions:

Bio 28 (August 19th)

Click-Chemistry and Cell Binding

Bio 29 (September 1st)

June 1st - Bio 1

Bio 30 (September 12th)

Bio 31 (September 13th)

Goal: Clicking oligonucleotides to carbohydrates and investigating if gel purification is a suitable method to distinguish clicked from unclicked oligonucleotides.

Hypothesis: Unclicked oligos should show a higher gel mobility than clicked ones, because they have the smaller size. This should be visible by a shift occurring between the concerend gel bands.

Material and methods: Before clicking carbohydrates to the designated oligonucleotides, click reactions were first tested with random DNA sequences ("Clickoligo A6") and the carbohydrate 2 - Azidoethyl alpha - Mannopyranoside. For detailed information about the click solution see the protocol below.

Detailed Protocol : Baseclick Protocol

June 2nd - Bio 2

20% PAGE Gel in 15ml - Gel 1
Goal:
Seperate the clicked oligos and the non-clicked one in order to see if the sugar binding to the DNA has worked.

Detailed Protocols: 20% native PAGE Gel in 15 ml, Sample preparation-Protocol-Gel 1

Material and methods: 4 samples were prepared: One control, which contained DNA diluted in a 1:10 ratio, and three DNA samples varying in their DNA concentration.

 

 

Results:

Discussion:

The wide and intense bands of the gel suggest a overloading of the gel with the DNA samples. This prohibits to separate the bands and to see any differences in their length.--> Reduce sample concentration for the next Gel Marker BIOLABS Low molecular weight suits better for our sample.

 

20% PAGE Gel in 15ml - Gel 2

Material and methods:

Second Gel preparation analogous to protocol listed above execpt the used DNA samples. Instead of using the previous sample we used a serial diluton with the clicked DNA samples in order to avoid overloading of the gel like it previously happened.

 

Gel time: 90 min at 150 V.

 

DNA samples have been diluted to reduce the DNA concentration in the gel. The serial dilution can be looked up in the protocol below.

 

Detailed protocols:

Sample preparation-Protocol-Gel 2

 

Result:

Discussion:

With the serial dilution it is possible to see a shift between the bands of the control sample and the clicked oligonucleotides. The intensity of the signal also reduces with the concentration. As the clicked oligonucleotides are heavier than the unclicked strands, the clicked DNA should lower mobility than the control sample. Additional bands are observed at 250 bp in the control as well as in the product samples. The reason for these bands could be a contamination of the unclicked oligonucelotides.

First Origami Folding:

June 11th - Bio 3

Goal: Determining the folding efficiency of the designed structure and the folding behaviour under varying MgCl2 concentrations.

Material and methods: Six samples were prepared. All samples had the following reagents in common but varyied in their MgCl2 concentration.

The samples originated from a stock solutions that had been prepared earlier. For detailed information concerning the preparation of the stock solution see the protocol below.

The MgCl2 concentration was increased from 10 mM to 20 mM in intervals of 2 mM as listed below. The samples were stocked up to 50µL with ddH2O.

The folding process was completed using a temperature ramp in a Thermo cycler. The ramp is depicted below:

 

Standard temperature ramp:

  1. Heat up to 65 °C and maintain the temperature for 15 min.
  2. Cool to 64 °C and maintain the temperature for 5 min.
  3. Go back to step 2 and reduce the temperature in increment of 1 °C four times.
  4. Maintain the temperature at 59 °C for 1h.
  5. Go to step 4 and reduce the temperature in increments of 1 °C 18 times.
  6. Maintain the temperature at $39^\circ C$ for 30 min.
  7. Go to step 6 and reduce the temperature in increments of 1 °C twice.
  8. Maintain the temperature at 36 °C for 5 min.
  9. Go to step 8 and reduce the temperature in increments of 1 °C 30 times.
  10. Maintain the temperature at 4 °C permanently.

Detailed protocols: Preparing the stock solutions

June 15th - Bio 4

Goal: The folding of Bio 3 was assayed by 2% agarose gel applying a voltage of 70V and subsequent TEM imaging in order to purify the folding products and to estimate the folding efficiency at different MgCl2 concentrations.

Material and methods: For detailed information concerning the preparation of the 2% agarose gel see the protocol below.

Detailed protocol: 2% Agarose Gel

 

Result:

Discussion:

The gel was stained with ethidium bromide. The electrophoresis results in two clearly separated gelbands at each magnesium concentration. All samples except the 20 mM sample are clearly visible. Moreover at every magnesium concentration 2 band can be observed.

 

As it is known individual origami structures exhibit a higher mobility than clustered structures (dimers, trimers etc.), therefore it can be assumed that the second bands (monomer bands) occuring in the gel contain the folded cylinder structures whereas the bands of lower mobility (dimer bands) contain clustered structures. Additionally the band of the scaffold is at the same height as the second bands at all magnesium concentrations which often occurs with ring structures. Anyway, this aspect has to be verified by TEM visualisation.

 

It is also noteworthy that all samples exhibit a considerable amout of aggregates in the gel pockets.

Croping the samples

 

Goal: Cutting of the bands at 10mM,12mM,14mM magnesium concentrations and subsequent purification via the Freeze N'Squeeze method (BioRad).

June 15th - Bio 5

Goal: Staining the samples of Bio 3 for TEM visualisation.

Material and methods:

Detailed protocol:

2% uranyl formate stain stock solution preparation

  • Boil ddH2O, keep boiling for 2-3 min to de-oxygenate
  • Weigh out 0.1 g uranyl formate in 15 mL falcon tube
  • Add 5 mL of still hot ddH2O to the falcon tube with the uranyl formate
  • Tightly close lid, wrap in aluminium foil and shake or vortex vigorously for 10 min
    • If the uranyl formate powder was rather fresh, you should obtain a turbid, yellowish solution
    • As the uranyl formate ages, the solutions typically appear more brownish
  • Filter solution using 0.22 μm syringe filter into a fresh falcon tube
  • Aliquot (200 µL aliquots into 1.5 mL tubes)
  • Centrifuge the aliquots at max speed for 5 min in a table top centrifuge
  • Wrap the aliquots in aluminium foil and store in freezer at -20 °C

Before staining

  • Thaw one aliquot
  • Add 1 μL 5 M NaOH to the tube wall at the surface of the liquid
  • vortex immediately for 2-3 min
  • Centrifuge at max speed for 3min in a table top centrifuge
  • Wrap in aluminium foil and store in fridge at 4 °

Grid preparation

  • Grids are placed on glass slide wrapped in parafilm and glow-discharged for 60s at 240V

Sample preparation

  • Pipette 3 µL of sample on grid surface and wait at least 60 s
    • Wait longer (3-4 min) for samples of lower concentration (eg. gel purified samples at about 1nM)
  • Meanwhile, for each sample, place 2 droplets of uranyl formate (7-10 µL each) on parafilm
  • dab excess liquid onto filter paper
  • briefly submerge grid into the first droplet and dab excess liquid immediately onto filter paper
  • submerge grid 20 s into the second droplet and dab excess liquid onto filter paper
  • allow grid to air-dry for at least 30min before imaging

 

Results:

TEM Pictures of Grid 14bot (Magnesium concentration 14 mM, bottom = second band of the Gel)

TEM Picture of Grid 12top (Magnesium concentration 12 mM, top = first band of the Gel)

Discussion:

Unexpectedly the band with MgCl2 concentration of 10 mM, which was of the strongest intensity in the gel, had a very low yield compared to the other two samples. Only a few structures were found on the TEM-grid. Dimers and clustered material were found in the dimer bands as previously assumed. As observed with the TEM, the samples with 14 mM magnesium concentration had the highest yield. This became the standard sample (S14) of the project. In future experiments S14 is going to be used as reference to compare it with samples that differ from it in their composition.

 

In general, all samples only contained a low amount of structures which highly likely originates from the effects of aggregation observed before. However, experimental issues like imprecise cutting of the gelbands could have impacted the results.

 

In summary, the interpretation of the gel's results was partially correct. It was possible to establish a presumption concerning the bands representing properly folded structures - the monomer bands. TEM visualisation was necessary to confirm this assumption and to learn more about the optimal MgCl2 concentration as the gel provided ambiguous results. Formation of the desired cylinders was most efficient for folding at 14 mM MgCl2 concentration. The following experiments were performed under these folding conditions. A scaffold sample is also applied to each gel in further pursued experiments, because it acts as a control.

Optimisation of the folding yield

June 17th - Bio 6

Goal: Motivated by the previous results, a first approach to prevent the aggregation in the gel pockets and thus improve the yield was to vary the concentration of the connector staples and the scaffold without changing the concentration ratio between scaffold and the other staples being 1:10. With this experiment the probability of possible cross linking between two structures should be reduced.

 

New folding stock at a constant MgCl2 concentration of 14mM.

 

Material and methods:

With:

The samples ran the standard temperature ramp. The folding was assayed by 1.5% agarose gel and subsequent TEM imaging.

 

Results:

1.5% agarose gel:

Discussion:

Comparable with the gel of Bio 4 this gel showed the same clear bands for each sample at 1.5 kb and slightly above 2.0 kb. Gel electrophoresis again results in monomer bands with the same gel mobility as the scaffold sample. The amount of aggregates has not improved as the gel still contained plenty of material in its pockets. On the contrary, aggregated folding products appeared to become more prevalent. It is conspicuous that lane (6) showed very weak bands, but it has to be considered that the HH sample contained less origami material compared to the samples of lane (4), (5). Thus, a diffuseness and lower intensity of the product band of lane (6) was expected. The product bands in lane (5) was of significantly greater intensity compared to the other bands. This generates the hypothesis that higher yield is obtained, but this can only be proven through TEM visualisation. On the other hand lane (5) led to fewer aggregates in the gel pocket than lane (4) which could have given rise to this higher intensity. Anyway, conclusive resolution of this issue required further analysis by TEM imaging.

 

TEM pictures of the CH-samples (Connector concentration half + normal scaffold concentration)

TEM pictures of the OC-samples (Without connector + normal scaffold concentration)

 

Dimers:

Monomers:

TEM pictures of HH-samples (Connector concentration half + half scaffold concentration)

Discussion:

The images gave rise to the assumption that the CH sample folded to higher yields. The TEM images showed a considerable quantity of ring structures. But observation at a higher resolution showed that the ring structures are not well folded but dented. Images showing upright cylinders also indicated the dented character of the cylinders as the helices are not orientated as parallel as they should be.

 

Compared to the CH samples the cylinders of the OC smaples show a more proper curvature. This indicates that the structures exhibit an high intrinsic curvature anyway and that the connector staples only close the cylinder and are not necessary to support curvature. The images show that the curvature is so high that the rings are partially interlocking.

 

The HH sample contained less DNA material, the yield was very low. The observed rings were closed and had a fairly proper curvature. Nevertheless, the problem with the aggregated material was also observed here.

 

Overall, the experiment does not show any improvement concerning aggregation in the gel pockets. Varying the connector staples' amount does not raise the folding yields, as it was the aim of this experiment. Regardless, it was an instructive experiment concerning the curvature. The experiment showed that the connector staples are not associated with the origamis' curvature as the structures are already highly curved without connectors. It appears that the connectors act as a kind of spacer between the two ends and let the ring form properly as they push the ends apart and finally close the ring.

June 18th - Bio 7

Goal: Determine the optimal annealing temperature of the origami structures by real time folding ("rt folding").

Material and methods: Six samples with increasing MgCl2 concentrations from 10 mM to 20mM and a scaffold sample were investigated.

The temperature ramp that was applied is depicted below.

Results: Plotting the data leads to the graphs below. The optimal annealing temperatures can be determined by the first derivation of the raw dataset as its maxima reveals the temperature points with the largest amount of produced dsDNA.

v

With the temperature's decrease, the production of dsDNA grew. This is understandable considering the fact that DNA is denatured at higher temperatures and thus the fluorescence signal which relies on double stranded DNA reduces. The first graph is the first derivative referring to the magnesium concentration of 10 mM which is the lowest MgCl2 concentration that was tested. It is sufficient to determine the maxima of this sample as the other samples show the same or part of these maxima. Optimal annealing times determined by rt folding were 42 °C, 45 °C, 48 °C and 51 °C. These temperatures were used to program new folding cycles at the Thermo cycler.

June 23rd - Bio 8

Goal: Based on results of the rt folding new individual folding programs on the Thermo Cycler were compiled and were supposed to help in optimizing the folding process. Thereby the three highest maxima of the 1st derivative (42°C, 45°C, 48°C, 51°C) were incorporated in three new programs.

Results: Three new folding programms

Bio 1

Bio 2

Bio 3

  1. 65.0° for 15:00
  2. 64.0° for 5:00 : -1° per cycle
  3. Go to 2, 13 times
  4. 51.0° 2:00:00
  5. 50.0° for 5:00 : -1° per cycle
  6. Go to 5, 2 times
  7. 48.0° 2:00:00
  8. 47.0° for 5:00 : -1° per cycle
  9. Go to 8, 5 times
  10. 42.0° 2:00:00
  11. 4.0° for ever
  1. 65.0° for 30:00
  2. 50.0° 2:00:00 : -1° per cycle
  3. Go to 2, 9 times
  4. 4.0° for ever
  1. 65.0° for 15:00
  2. 51.0° 2:00:00
  3. 48.0° 2:00:00
  4. 45.0° 2:00:00
  5. 42.0° 2:00:00
  6. 4.0° for ever

June 24th - Bio 9

Goal: The folding of Bio 8 was assayed by 2% agarose gel.

Results:

Roti-stain:

Discussion:

This gel was much less informative than the previous ones. All bands in the gel were barely visible and the programs appear to completely abolish productive folding as aggregated material heavily increased. The results are sufficient to conclusively say that the reason for aggregation does not mainly originate from the folding program. As the gel pockets contained a lot of material that has not been explored yet, the aggregates of the pockets were cut out of the gel and observed with the TEM. As was previously ascertained, the pockets contained unfolded material. No ring structures were found (see Bio 11).

June 26th - Bio 10

TEM visualisation of the aggregates:

June 29th - Bio 11

Goal: Folding sufficient amount of origami structures for subsequent cell medium stability tests. Additionaly, fodling origami structures without connector staples to see if subsequent addition of the connector staples improves the aggregation problem.

Material and methods:

Sample composition for stability tests (200µL - Fourfold amount of HH sample; see 17th June):

HH Sample

Sample composition without connectors (100µL - double amount of the OC sample; see 17th June):

OC1 sample

June 30th - Bio 12

Goal: 1.5% agarose gel for Bio 11

Material and methods:

Detailed protocol:

Changes: 2,25g Agarose in 165ml TAE-buffer without MgCl2 Gel running: 60V Start time: 11:40 End time: 14:20

Sample preparation for agarose gel:

Scaffold

HH

OC1

  • 6µL 1kb lad
  • 9µL H20
  • 1µL Scaffold p7560

=12µL

  • 200µL HH
  • 40µL lb

 

=240µL

  • 100µL OC1
  • 20µL lb

 

=120µL

lb: loading buffer

1kb lad: 1kb ladder

 

Results:

Stained with Ethidium Bromid:

Discussion: As the gel bands were hardly visible, Bio 11/12 has to be repeated.

July 2nd - Bio 13

Motivation: Retry Bio 11.

Goal: Folding sufficient amount of origami structures for subsequent cell medium stability tests. Additionaly, fodling origami structures without connector staples to see if subsequent addition of the connector staples improves the aggregation problem.

Material and methods: As additional references a standard sample S14, OC2 (without connectors, sugars and fluorophores) and MC (with connectors) samples are folded and assayed with a 1% agarose gel.

Sample composition for stability tests (200µL - Fourfold amount of HH sample; see 17th June):

 

HH sample

Sample composition without connectors (100µL - double amount of the OC sample; see 17th June):

 

OC1 sample

References:

MC sample (50µL):

OC2 sample (50µL):

Standard S14 (50µL, 14 mM MgCl2 concentration):

Results: due to a pippeting mistake the probe OC1 was lost.

July 3rd - Bio 14

 

Discussion: The band MC displayed the best folding yield. This provides first hints that subsequent addition of fluorophore and sugar staples significantly improve the yield. It is not possible to compare the band with OC1 due to error during sample loading.

July 9th - Bio 15

Top: (1) ladder, (2) scaffold, (3) S14, (4) Z RT, (5) F RT, (6) B RT, (7) C2 RT, (8) Z 37°, (9) F 37°, (10) B 37°, (11) C2 37°, (12) Z 45°

 

Bottom:(1) ladder, (2) F 45°, (3) B 45°, (4) C2 45°, (5) Z 41°, (6) F 41°, (7) B 41°, (8) C2 41°

July 10th - Bio 16

(pocket 4 "empty" because mistake during loading the gel. Despite of this, there was material which trsnfered from 3 to pocket 4)

July 15th/16th - Bio 17

Sample preparation for dye labeling (see here)

July 20th - Bio 18

Goal: Testing if the subsequently added fluorescent staples are incorporate into the origami structure. First try with Aminoallyl-dUTP-XX - ATTO-488 form Jena Biosicence with a laser scanner.

The gel contains origami structures that are labeled with fluorescent dyes with 1:10 and 1:100 dilution.

The gel is loaded with 7 µl ladder, 60 µl origami sample and 1.25 µl dye.

Disscusion: The gel is clearly overexposed and the sample are too concentrated. But staple and origami exhibit a clear separation in both lanes. This provides first hints for a successful incorporation.

July 22nd - Bio 19

Sample preparation for dye labeling

July 23rd - Bio 20

Repetition of the magnesium concentration screening.

Goal: Finding the ideal magnesium concentration for folding when adding the functional staples subsequently.

1.5% Agarose at 70 V for 150 min

Discussion: Analyzation of the fluorescent intensity showed that a concentration of 14 mM magnesium has the best ratio of aggregation to folded origami.

July 24th - Bio 21

Fluorescence gel 2 - etbr stained

Goal: Verificaton of the incorporation of the fluorescent staples in structure. Determination of the ideal excess of flurescent staples. A serial dilution of the structure was made to avoid overexitazion when visualizing the gel.

The staples where labed with Aminoallyl-dUTP-XX - ATTO-488 form Jena Bioscience as described in Bio 19.

1.5% agarose gel without staining loaded with 50 µl sample at 70 V for 150 min.

Gel after exitation with laser wavelength of 500 nm.

V: Dilution, Ü: Excess

Ü1 is a 10 times, Ü2 a 5 times and Ü3 a 3 times excess of the fluorophore staples. The fluorescent and sugar staples were added after folding at room temperature.

Fluorescence gel 2

Gel under UV light.

Discussion: The pictures were taken with a laser at a wavelength of 500 nm before staining the gel. The first picture shows the same gel after staining with EtBr. The bands of both the stained and the unstained gel are consistent and is therefore a proof for the incorporation of the fluorophore staples into the folded origami structure. To exclude the possibility of spectral crosstalk, the unstained gel was also visualized with UV light. There where no bands visible.

 

File: measure.pdf

July 27th - Bio 22

Goal: Verification of the magnesium screening, due to the elevated room temperatue during the folding procces of Bio 20.

Discussion: Here the fluorophore and sugar staples where added after completion of the folding procces at room temperature in accordance to Bio 20. By analyzing the fluorescence intensity and comparing the amount of folded origami to the amount of aggregation, it was confirmed once more that 14 mM magnesium concentration is ideal for our structure.

July 30th - Bio 23

Goal: Testing if lowering the origami concentration in the folding proccess reduces the percentage of aggregates.

Subsequent addition:

Discussion: Analysis of the fluorescent intensity showed no decrease of aggregation for lower concentrations.

Stability tests:

August 4th - Bio 24

Goal 1: Testing the stability of the structure in cellular enviroment.

--> Stability test 1. Variation of serum + origami ration and incubation time.

Material:

  • 800 µl origami stock
  • serum: FCS (fetal calf serum)

Procedure:

  1. Determining the concentration of the 800 µl - origami solution with the nanodrop (2.3 ng/µl --> 0.46 nM)
  2. Testing two different origami:serum ratios --> 1:1 (1) as well as 4:1 (2)
  3. Testing four different incubation times: 0.3 h, 1 h, 2 h, 6 h
  1. Reference: 50 µl origami without serum
  2. Mix samples with serum.
  3. After 0.3 h remove 5 µl from the tube with the higher origami concentration and freeze it immediately in order to estimate later if the Amicon filtration had damaged the structures via TEM observation.
  4. Use the 100K Amicon filters. Centrifugate with 7000 rpm for 7 min. Repeat this step 4 times.
  5. Repeat step 3/4 after all incubation times.
  6. After centrifugation, freeze the sample immediately.

 

Goal 2: Preparing new origami stock for further stability tests

Procedure: Prepare 8 x 100 µl stock solution as listed below.

Subsequent addition (at 25 °C):

August 5th - Bio 25

Goal: Gel for stability test 1 - Bio 24 (see 4th August)

Note:

  • Reference (origami naked) did not incubate 6 h but was immediately put into the gel.
  • After running, the gel (1.5% Agarose-Gel) was stained with Sybr Gold (100 ml TAE + 11 mM MgCl2).

August 6th - Bio 26

Goal 1: Purification of the prepared 800 µl stock of Bio 33 (see 4th August)

Goal 2: TEM observation of the non-centrifugated samples of Bio 32.

Procedure: Staining with uranyl format (see protocol)

Discussion:

The images show the TEM observation of the origami solution incubated for 0.3 h in FCS. The origami+serum ratio was 4:1.

As the proteins were not removed from the sample it was not possible to find any structures on the TEM grids (neither on the grids with the other samples (1 h, 2 h, 6 h).

August 11th - Bio 27

Goal: Stability test 2 in cell serum (FCS) without Amicon-method but instant gel loading

Procedure:

  • Incubation times: 0.3 h, 1 h, 2 h, 6 h
  • Ratios: 1:1, 4:1
  • References: Origami naked which was at RT for 6 h + scaffold.
  • Let serum+origami incubate at RT (as the RT on this day was quite high --> Thermo cycler at 25 °C)
  • Prepare 9 Eppendorf tubes with 25 µL origami (800 µl stock solution --> at the end of the experiment the origami samples should have been at RT for the same time period to create the most comparable reaction conditions as possible) and start adding 25 µL (for a 1:1 ratio) to the first Eppendorf tube and 8.3 µL (for a 4:1 ratio). This two samples are going to be the one that incubate the longest (6 h).
  • After 4 h add the same amounts of serum to the next two eppis.
  • After 5 h again add serum to the next samples and simultaneously start to prepare the 1.5% agarose gel.
  • After 5.5 h add serum to the last two samples. These are the one which only incubate half a hour in the FCS.
  • If the 6 h have passed immediately load the gel with the samples.

 

Discussion:

  • As the gel bands were comparable to the first gel concerning their intensity and compactness four samples (the one corresponding to the 4:1 ratio) were cut out off the gel.
  • TEM observation is expected to provide further information about the structures.

August 19th - Bio 28

Further approaches to avoid aggregations in gel pockets:

  • Based on previous experiments fluorophore staples are problematic --> Varying the concentration of the fluorophore staples --> 5x excess instead of the standard 10x excess.
  • "Isothermal" temperature ramp: Breaking all bonds in the origami solution by applying a temperature of 80 °C for 5 min and instantly cool down to 50 °C (this is an example --> crucial interval is 46 °C-52 °C) and maintain this temperature for 18 h.
  • In genereal it would be worth trying to expand the temperature ramp to 72 h total running time.
  • There are some problematic points in the design --> leave away the corresponding staples in order to see if this leads to less aggregation.

Clickchemistry and Cell binding:

September 1st - Bio 29

Goal: Preparing the stock solutions (carbohydrates and master mix) for clicking sugar molecules to the modified click oligonucleotides

Material and methods:

Available carbohydrates (dried):

Dissolve sugar in ddH2O to 10 mM solution

Calculation:

5 mg / x g/mol = y mol

ci = ni/Vi → V=ni/ci

e.g. 8.136·10 -6 mol / (10·10 -3 mol/L) = 813.6 µL

Mastermix Click Oligos

Click-oligonucleotides 1 - 24 → 10 µL per Click Oligo → 240 µL in 100 µM Solution Clicking act upon the Oligo-Click-M user manual provided from baseclick:

10 µL DNA with 20 µL carbohydrate → 1 nmol DNA dissolved in 1 mL ddH2O → 1 µM

September 12th - Bio 30

Goal: Clicking carbohydrates to the modified click oligonucleotides

Material and methods:

→ Use the master mixes prepared in Bio 29. → 10 µL mastermix with 20 µL of each type of carbohydrate. The click reaction was performed using baseclick's Oligo-Click-M kit.

September 13th - Bio 31

Goal: Prepare 1.6 mL origami solution for fluorescence imaging

Material and methods:

Formulation for 100 µL origami solution:

For 1.6mL formulation 16x 100 µL origami solution is necessary → 76.8 µL fluorophore staples are required which must be enzymatically transferred before with the terminal transferase kit.

76.8 µL / 9.6 µL = 8

Required material for the terminal transferase:

→ 1 h in Thermo cycler maintaining 37 °C

Add

  • 20 µL NaAC (3 M)
  • 460 µL 100% Ethanol

All these steps were performed in two 2 mL Eppendorf tubes.

After 1 h Thermo Cycler the enzymatic reaction is stopped by putting the samples at -20 °C for 1 h.

4 samples were prepared. Each in duplicates:

Negative control: Standard origami stock solution. Subsequent addition of 4.8 µL unclicked sugar staples.

Positive control: Standard origami stock solution. Subsequent addition of 4.8 µL unclicked sugar staples. For the subsequent experiment this controle will be incubeated with lipofectamin to versilitate unspicific uptake.

SLex: Standard origami stock solution. Subsequent addition of 0.5 µL clicked sugar staples.

Tri-LN: Standard origami stock solution. Subsequent addition of 0.4 µL clicked sugar staples.

Add 4.8 µL transferred fluorophores to each sample. Store the samples at RT.

September 23th - Bio 32

Goal: 1% agarose gel for Bio 35

Material and methods:

200 µL for each gel pocket needs modification of the gel comb by taping some pockets to one big pocket.

Transfer samples in 1.5 mL Eppendorf tubes.

For 200 µL sample, 33.4 µL loading buffer was necessary.

All samples were cut out the gel for imaging with the fluorescence microscope.

 

Results:

First gel:

After 3 h the gel was stained with Etbr for 45 min and washed in TAE with MgCl2 for 10min.

Second gel:

The second gel was immediately stained with Roti Gelstain.

September 24th - Bio 33

Goal: Preparing the samples (Bio 36) for fluorescence microscopy

Material and methods:

Final volume: 90 µL The final concentration is the same for each sample.

Detailed protocol:

A549 cells were provided by the cell culture supervisor. The cells where incubated with the origami cell medium mixture for 20 min at 37 °C in 5% CO2. The cells were washed 3 times with 1 mL cold PBS. For the microscopy 90 µL of L-15 medium where added. The overview scans were taken with Nikon TI Eclipse at 100x magnification. The exposure time for the Cy5 exitation was 200 ms, laser power 100%, binning 1 and signal to noise ratio 10 MHz. For this measurements the Cy5 laser and filter sets where used (Excitation 628 nm with Bandwidth 40 nm and Emission 692 nm with Bandwidth 40 nm). The brightfield pictures where take at a exposure time of 20 ms and otherwise the same settings. All pictures where taken under the same conditions and are therefore comparable.

 

Results:

Neg 1:

Neg 2:

SLex 1:

SLex 2:

Tri-LN 1:

Tri_LN 2:

Discussion: The fluorescence signal’s intensity was evaluated by measuring 6 proportionate areas within the corresponding scans. This data does not show any difference in the fluorescence intensity between the negative sample and the SLex coated origami structure. On the other hand that A549 cells exhibit a higher affinity to Tri-LN. Also a high amount of photobleaching was observed. In conclusion a different fluorescent dye should be used for subsequent experiments.

September 28th - Bio 34

Goal: Repeat Bio 33 but with cell mask staining. This should prove if the ormigami is located at the cell membran.

Material and methods: The cell membrane staining was perfomed following the protocol from Fisher Thermo Scientific: https://www.thermofisher.com/order/catalog/product/C10045. Otherwise the experiment was perfomed in accordance to Bio 33. Note here thar the single snap shots where taken at different exposure times and noise ratios and are therefore not comparable. The overview scans where taken at the same settings as in Bio 33. The excitation time was chosen to be 30 ms for the cell mask excitation. The following filter set and laser was used for the cell mask pictures : eGFP (Excitation 470 nm with Bandwidth 40 nm and Emission 525 nm with Bandwidth 50 nm)

Medium:

Sample preparation:

Sample preparation:

SLex 1:

SLex 2:

Discussion: The pictures with the lower contrast are the pictures taken at 628 nm and correspond to the origami. All of the shown picture series exhibit one distinct spot at all three modes: Cy5 excitation, eGFP excitation and brightfield image. One can see a clear colocalization of the Cy5 and eGFP channel. It also correlates with the cell membrane seen in the brighfield pictures. Most of the observed cells where dead. This is maybe due to a to slow handling for the microscopy preparation. For future experiments to avoid cell dead optimization during the proccess while taking the pictures are necessary. But all in all this provides first hints that the origami is bound to the cell membrane.