Biomod/2014/Kashiwa/Experiments

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EXPERIMENTS

 Highlights


The agarose gel electrophoresis.


Fig.1-1(a)-2. TEM image of the Wall.

Fig.1-1(a)-2. TEM image of the Wall.


Fig.1-1(a)-2. TEM image of the Wall.

Fig.1-1(a)-2. TEM image of the Wall.





 The Receptor

1-1. Preparing the components

Three componets were prepared to develop the Receptor: The Wall of DNA origami, MISTIC for embedding into the liposome and the Activator. Please check our Design page to check each component's role.

1-1(a). Folding the Wall

Fig.1-1(a)-1. Gel analysis of the Walls annealed in different time.

Fig.1-1(a)-2. Gel analysis of the Walls annealed in different temperature.

In this experiment, the assembly condition of the Wall structure was optimized and results were analyzed by agarose gel electrophoresis. The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.


Fig.1-1(a)-3. Gel analysis of the Walls annealed in different concentration of NaCl.

Fig.1-1(a)-4. Gel analysis of the Walls annealed in different concentration of MgCl2.

The optimum results were following:

  • Optimum concentration of MgCl2 is 15 mM.
  • Optimum temperature of annealing is 45.3 °C.
  • Optimum time of annealing is 5 hours.
  • Optimum concentration of NaCl is 2.5 mM.



Fig.1-1(a)-5. TEM image of the Wall.

The folding is corroborated by the TEM image (Fig.1-1-1.).


1-1(b). Producing MISTIC

We introduced the variant to the gene of MISTIC, a protein which penetrates the membrane for 4 times, by using Quick Change method. The purpose of this experiment is to produce mono-cysteine MISTIC. By this operation, we would be able to label arbitrary position of MISTIC when we want to label cysteine. We introduced variation to valine which was at 29th from the initiation codon.

The wild type of the configuration of MISTIC was as follows.

MFCTFFEKHHRKWDILLEKSTGVMEAMKVTSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQ LDDDTAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKE

Underlined parts are considered to be the α-helix structures which penetrate membranes. A red letter show the part which we introduced variation in this experiment.

We confirmed the introduction of variant by sequencing.

(i) Preparation of templateDNA of MISTIC for PUREfrex

In order to express MISTIC using protein synthesis using recombinant elements (PURE) frex, we introduced T7 promoter, SD sequence and T7 terminator to the variant DNA which we made. This introduction was conducted by PCR. PURE frex is a reconstructed cell-free system for transcription and translation reaction. At the same time, we substituted valine for cysteine3 and completed to make mono-cysteine configuration. We introduced Histag to the C-terminus and replaced Phe2 by amber, one of a termination codon. The latter introduction was done to introduce biotin to the N-terminus using unnatural amino acids.

Considering the efficiency of inserting unnatural amino acids would be influenced by the position of insertion, we made seven types of DNA and used the optimum replacement type of Phe2 to amber. Final configuration of MISTIC is as follows.

M+VTFFEKHHRKWDILLEKSTGVMEAMKCTSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQLDDD TAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKEHHHHHH

+ is an abbreviation for unnatural amino acid.

Fig.1-1(b)-1. Agarose gel electrophoresis of PCR products.

The completion of DNA for PURE frex was confirmed by gel electrophoresis. If the existence of the DNA which the length was the same as the one which we wanted was recognized, we resulted that the DNA was completed.

As a single band was observed at the position we aimed (455bp), we resulted that DNA for PURE frex was completed.


(ii) Expression and purification of MISTIC protein

Fig.1-1(b)-2. SDS-PAGE of PURE products.

We added MISTIC gene to the solution of PURE frex, incubated at 37&dig;C for 2 hours and formed MISTIC protein. The expression in PURE frex was confirmed by SDS-PAGE. In order to distinguish between the factor of PURE flex and MISTIC, we added 35S-Met to PURE flex and labelled MISTIC with a radioisotope and detected it by Photostimulated luminescence (PSL).

As we could observe the band at the aimed position (13kDa), we considered that MISTIC was formed in PURE flex.


Fig.1-1(b)-3. SDS-PAGE of PURE products and Purification Products.

We then purified a product of PURE flex using Histag column. The confirmation of the purification was done by SDS-PAGE.

As we could see MISTIC was included to the division of elusion and impurities were few, we resulted that the purification was succeeded.


(iii) Confirmation of the introduction of unnatural amino acids

Fig.1-1(b)-4. SDS-PAGE of PURE products

By confirming that MISTIC forms when we used unnatural amino acids and cannot form without them, we would assay that that unnatural amino acids are included in MISTIC. We detected MISTIC labelled by a radioisotope by PSL (same method stated in (iii)).

As we could see that MISTIC is not formed when unnatural amino acids are not added, we could determine that tRNA of amino acids which are inserted to the codon of amber competitively with RF1 does not exist. From this fact, we could confirm that unnatural amino acids are included in MISTIC including amber.


(iv) Confirmation of the ability of access to biotin under the non-denatured condition

Fig.1-1(b)-5. SDS-PAGE of PURE products

By certifying whether or not MISTIC expressed in PURE frex are removed by the magnetic beads which streptavidin (SA) is connected, we confirmed that SA can access to niotin under the non-denatured condition. To exclude the influence of other PURE factors, we labelled it by 35S-Met and assayed by PSL.

We loaded same amount of the product of PURE to all lanes. As only the amount of MISTIC with biotin is decreased, biotin connected to MISTIC is able to be accessed and MISTIC was removed from solution through that connection.


(v) Confirmation of modification of MISTIC to cysteine

Fig.1-1(b)-6. SDS-PAGE of modified mistic (Cy3/SYPRO ORENGE)

MISTIC was labelled by Cy3 maleimide (Cy3-MA) and confirmed whether cysteine residue of MISTIC is able to be modified or not. 0.45µL of 11mM Cy3-MA and 50µL of 3.26µM MISTIC were mixed and incubated for 30 minutes. Products were perdormed to electrophoresis using SDS-PAGE ageter purification and fluorescence was observed. After that, protein was dyed by SYPRO ORANGE and fluorescence was observed again.

As we could see the fluorescence of Cy3 at the position of the band of MISTIC, the modification seemed to be successful. Comparing the intensity of fluorescence with other lanes which we put the same amount of Cy3-MA, the modification rate is presumed to be --%.

1-1(c). Designing the Activator

Two restriction enzymes, HindⅢ and Lambda Exonuclease, were compared to choose which is appropriate for the Activator. Two aspects were evaluated in this experiment: modification with oligonucleases and enzyme activity.

(i) Evaluating modification with oligonucleatides

Fig.1-1(c)-1. BS(PEG)9 for oligo-modification.

Hind&8546; and Lambda Exonuclease are modified with oligonucleotides to join the Activator and the Anchor. BS(PEG)9 was used for the modification.


On HindⅢ, the modification was analyzed by Native-PAGE. Oligonucleotides are labelled with Cy3 and HindⅢ is labelled with SYBR GREEN to confirm the modification.

Fig.1-1(c)-2. Cy3 fluorescence image of oligo-modified HindⅢ by Native-PAGE.

Fig.1-1(c)-3. SYPRO-ORANGE fluorescence image of oligo-modified HindⅢ by Native-PAGE.


In the both figures, a band was observed in the same position in lane ○○. The modification of Hind3 with Cy3-oligonucleotides was therefore confirmed.

On Lamda Exonuclease, the modification was confirmed by Native-PAGE in the same way as HindⅢ.

Fig.1-1(c)-4. Cy3 fluorescence image of oligo-modified Lamda Exonuclease by Native-PAGE.

Fig.1-1(c)-5. SYPRO-ORANGE fluorescence image of oligo-modified Lamda Exonuclease by Native-PAGE.


The result shows Lamda Exonuclease was modified with oligonucleotide successfully.

(ii) Evaluating enzyme activity

Fig.1-1(c)-7. DNA cleavage reaction by Lamda Exonuclease.

Fig.1-1(c)-6. DNA cleavage reaction by HindⅢ.

HindⅢ is an restriction endonuclease that recognizes base sequence 5’-AAGCTT-3’ and cleaves it, while Lambda Exonuclease degrades one strand from 5'-phosphoryl termini of dsDNA.


In this experiment, enzyme activity of HindⅢ and Lambda Exonuclease was evaluated in various conditions. The purpose is to confirm that HindⅢ and Lambda Exonuclease have activity in condition for DNA origami. The activity was analyzed by Native-PAGE.

Fig.1-1(c)-9. Native-PAGE shows Lamda activity depending on PH.

Fig.1-1(c)-8. Native-PAGE shows HindⅢ activity depending on PH.

First, enzyme activity depending on PH was analyzed.

The result showed that Lambda Exonuclease can act in pH7.5-8.5. In pH9.0 and pH9.4, dsDNA seems to be unstable. So, pH7.5-8.5 is appropriate for reaction of Lambda Exonuclease and DNA.


Fig.1-1(c)-11. Native-PAGE shows Lamda activity depending on concentration of MgCl2.

Fig.1-1(c)-10. Native-PAGE shows HindⅢ activity depending on concentration of MgCl2.

Then enzyme activity depending on concentration of MgCl2 was analyzed.

The result showed that Lambda Exonuclease can act in mM MgCl2.


1-2. Embedding the Wall into the liposome

Embedding the Wall into the liposome needs two steps: modifying the Wall with cholesterol and putting them into the liposome. The experiments were done individually.

(i) Hybridization of cholesterol oligomer with the Walls

In this experiment, we examined the best concentration and the number of staples which the cholesterol oligomers can connect. Hybridization of cholesterol oligomer with the Walls is needed to penetrate the Walls into the membrane of liposomes. The result was assayed by 1% agarose gel electrophoresis.

Fig.1-2-1. Confocal microscope image of GUV including Walls.

If the cholesterol oligomer was connected to the Walls the band would be smear. In sample 2_1~4, 3_1, 3_2, 5_1, 5_2, 6_1. Considering that sample 2 and 3 do not have staple for hybridization, the Walls of sample 2 and 3 aggregated by the existence of cholesterol oligomer. Sample 5_1, 5_2 and 6_1 are considered to be successful in hybridization. As the band of sample 5_2 and 6_1 are weaker than that of sample 5_1, we decided that the best condition was sample5_1.


Fig.1-2-1. Confocal microscope image of GUV including Walls.

In this experiment, the Wall which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV) to penetrate the liposome from inside. Inclusion of Motor-Monomers and Walls was observed by confocal microscope.


The connection of Q-dot to the Wall was confirmed by agarose gel electrophoresis.

Fig.1-2-2. Agarose gel electrophoresis showing the existence of Q-dots.

Fig.1-2-3. Agarose gel electrophoresis showing the existence of DNA.


The first picture shows the existence of Q-dots and the second picture shows the existence of DNA.

Comparing the seventh, eighth and ninth lane from the left in the first picture, we can see the difference in the position of the Q-dots. Comparing the eighth and eighth lane from the left in the two pictures, we can see the position of DNA, in this case, Walls, and the Q-dots in the same position. This data shows that the Q-dots connected to the Walls through the connection of biotin and streptavidin.

1-3. Linking the Activator to the liposome

1-4. Combining the Wall and the Activator

1-5. Separating the Wall from the Activator




 The Motor

2-1. Producing the Motor-Monomer

Producing the Motor-Monomer is divided into three sections: Folding the Motor-Monomer body, synthesizing divalent streptavidin and equipping the Motor-Monomer body with divalent streptavidin.

2-1(a). Folding the Motor-Monomer body

Fig.2-1(a)-1. Gel analysis of the Monomers annealed in different time.

Fig.2-1(a)-2. Gel analysis of the Monomers annealed in different temperature.

In this experiment, the assembly condition of the Motor-Monomer structure was optimized and results were analyzed by agarose gel electrophoresis. The optimum conditions were confirmed by comparing migration distances of each samples. The sample of which migration distance is the longest was regarded as the optimum condition.


Fig.2-1(a)-3. Gel analysis of the Monomers annealed in different concentration of NaCl.

Fig.2-1(a)-4. Gel analysis of the Monomers annealed in different concentration of MgCl2.

The optimum results were following:

  • Optimum concentration of MgCl2 is 15 mM.
  • Optimum temperature of annealing is 45.3 °C.
  • Optimum time of annealing is 5 hours.
  • Optimum concentration of NaCl is 2.5 mM.


Fig.2-1(a)-5. TEM image for the Motor-Monomers.

The folding is corroborated by the TEM image.


2-1(b). Synthesizing divalent streptavidin

Fig.2-1(b)-1. SDS-PAGE showing purified divalent SA.

In this experiment, divalent streptavidin (SA) was synthesized for equipment to the Motor-Monomers.

TaKaRa Competent Cell BL21 was used for protein expression. The purification of divalent SA on the nickel-affinity column was analyzed by SDS-PAGE.


Fig.2-1(b)-2. SDS-PAGE showing the affinity of divalent SA with biotin-modified oligos.

The affinity of divalent SA with biotin-modified oligonucleotides was then analyzed by SDS-PAGE.

2-1(c). Equipping the Motor-Monomer with SA

Because linking the Motor-Monomer and divalent SA uses modification of divalent SA by NHS ester reaction and click reaction, SA was linked with cy5-modified oligonucleotide via click reaction and NHS ester reaction, then the ability of modified SA to bind biotin-nucleotide was confirmed in this experiment.

Fig.2-1-3. .

Fig.2-1-4. .


Figure 2-1-3 shows that SA was linked with cy5-modified oligonucleotide via click reaction. In Fig.2-1-4, because the lane of only Cy5 and NH2-modified oligonucleotide is not stained, the stained bands is from biotin-oligonucleotide. This picture shows modified SA can bind biotin. It may show the ability to bind biotin decreases as the amount of modification of SA increases, but the experiment of modification SA with Cy5-NHS deny. The reason of that may be derived from steric hindrance of Cy5-oligonucleotide which binds to SA.

These figures confirm the ability of modified SA to bind biotin-nucleotide.

2-2. Deactivating and activating the binding capacity of streptavidin

Fig.2-2-1. Mechanism of this experiment.

In this experiment, deactivation and reactivation of streptavidin (SA) were confirmed by Native PAGE.

First, two complementary oligonucleotides having biotin or desthiobiotin were annealed. The oligos having biotin is named Linker and the oligos having desthiobiotin is named Blocker. The resulting dsDNA was then mixed with divalent SA for deactivation. After removing excess SA with biotin-beads, ds-DNA bound to divalent SA was cut by HindⅢ for reactivation. Another oligonucleotides having biotin, named Chaser, were then added to replace Blocker.

Blocker was labeled with Cy3 and Chaser was labeled with Cy5 to confirm the replacement by Native-PAGE.


Fig.2-2-2. Cy3 fluorescence image of Native-PAGE.

Fig.2-2-3. Cy5 fluorescence image of Native-PAGE .


In fig.2-2-3, multiple bands of SA-dsDNA complexes appears in lane 3 which is caused by non-ideal complexes. It is considered that biotin-beads successfully distinguished the ideal SA-dsDNA complex because the upper band in lane 3 mostly disappeared in lane 4.

In fig.2-2-4, a band at the same position of the ideal SA-dsDNA complex is only shown in lane 7. We therefore confirmed the replacement of Blocker with Chaser after cutting dsDNA by HindⅢ and that the replacement did not happen without HindⅢ.

2-3. Putting the Motor-Monomers into the liposome

In this experiment, Motor-Monomers which have biotin staple combined with Q-dot connected with streptavidin were put into the giant unilamellar vesicles (GUV). Inclusion of Motor-Monomers was observed by confocal microscope.

GUVs were dyed by Nile Red. Yellow green dots indicate the fluorescence of Nile Red and red dots indicate the fluorescence of Q-dots combined with Motor-Monomers.

Fig.2-3-1. Confocal microscope image of GUV including Motor-Monomers.

Fig.2-1-2. Confocal microscope image of GUV not including Motor-Monomers.


Comparing the two pictures, we can see the red dots inside the GUV only in the picture showing the GUVs containing Motor-Monomers in the inner solution. From this fact, we could confirm the inclusion of Motor-Monomers into the GUV. As the excitation and fluorescence wave length of Nile Red and Q-dots were close, the membrane of GUV was shown in red even in the picture of GUV not including the Q-dots.


The connection of Q-dot to the Motor-Monomer was then confirmed by the agarose gel electrophoresis.

Fig.2-1-3. Agarose gel electrophoresis showing existence of Q-dots.

Fig.2-1-3. Agarose gel electrophoresis showing existence of DNA.


The first picture shows the existence of Q-dots and the second picture shows the existence of DNA. Comparing the fourth, fifth and sixth lane from the left in the first picture, we can see the difference in the position of the Q-dots. Comparing the fifth and sixth lane from the left in the two pictures, we can see the position of DNA, in this case, Motor-Monomers, and the Q-dots in the same position. This data shows that the Q-dots connected to the Motor-Monomers through the connection of biotin and streptavidin.


© 2014 UTokyo Chem & Bio

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