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<body> EXPERIMENTS

<a name="background"> 1. The Sensing System: The Receptor</a>

This section described the details of our approach to develop the sensing system "Receptor" of PoLICe. The approach mainly consists of four steps as follows.

<a href="#1-1">1-1. Preparation of the components </a>

<a href="#1-1-1">1-1(a). Folding of the Wall </a>
<a href="#1-1-2">1-1(b). Production of MISTIC </a>
<a href="#1-1-3">1-1(c). Design of the Activator </a>

<a href="#1-2">1-2. Embedment of the Wall into the liposome </a>
<a href="#1-3">1-3. Linkage of the Activator to the liposome </a>
<a href="#1-4">1-4. Control of the Wall conformation</a>

1-1. Preparation of the components

Three components were prepared to develop the Receptor: The Wall of DNA origami, MISTIC and the Activator. Please check our <a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design">Design page</a> to check each component's role.

1-1(a). Folding of the Wall

<img src="http://openwetware.org/images/9/99/Walltimeaneal.png" width="180px">	<img src="http://openwetware.org/images/2/29/Annealringase.png" width="180px">	<img src="http://openwetware.org/images/1/1c/Wallnaclkashiwa.png" width="180px">	<img src="http://openwetware.org/images/e/e0/Wallmgcl2l2.png" width="180px">
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.	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.

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)-5. TEM image for the Motor-Monomers.

The optimum results are as follows.

Concentration of MgCl₂ : 15 mM
Temperature of annealing : 45.3 °C
Time of annealing : 5 hours
Concentration of NaCl : 2.5 mM

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

1-1(b). Production of MISTIC

(i) Mistic gene mutation

We introduced the mutation to the MISTIC, a protein which penetrates the membrane for 4 times, by Quick Change method. We introduce Cys residue at the position after first α-herix for connection with enzyme or fluorescent labeling (29th valine to cysteine).

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

MFCTFFEKHHRKWDILLEKSTGVMEAMKVTSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQ LDDDTAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKE

We made 4 types of mutants (Cys3 to Val, Val29 to Cys, Cys between 84-85 and Cys between 110-111), but only used this one.

Underlined parts form α-helix structures which are considered to be the membranes penetrating domain.

A red letter shows the part which we introduced variation in this experiment.

We confirmed the introduction of mutation by sequencing.

(ii) Preparation of templateDNA of MISTIC for PUREfrex

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

In order to express MISTIC by PURE(protein synthesis using recombinant elements)frex, we added T7 promoter, SD sequence and T7 terminator to the mutatn DNA. At the same time, we substituted valine for Cys3 then completed to make mono-cysteine mutant. We also added Histag to the C-terminus and replaced some amino acids near N-terminus by amber, one of a termination codon, to introduce biotin to the N-terminus using unnatural amino acids.

We tried first seven amino acids of Mistic for biotin insertion, and found that position 3 is the best place.

M+VTFFEKHHRKWDILLEKSTGVMEAMKCTSEEKEQLSTAIDRMNEGLDAFIQLYNESEIDEPLIQLDDD TAELMKQARDMYGQEKLNEKLNTIIKQILSISVSEEGEKEHHHHHH

+ is an abbreviation for unnatural amino acid.

PURE frex is reconstructed cell-free system for transcription and translation reaction.

We checked the PCR product by agarose electrophoresis, and conformed that there are no extra band and the length is that of expected one (455bp).

(iii) Expression and purification of MISTIC protein

1. Expression

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

We added MISTIC gene to reaction solution, incubated at 37℃ for 2 hours and obtained MISTIC protein. The expression in PUREfrex was confirmed by SDS-PAGE. In order to distinguish between the factors of PUREfrex and MISTIC, we added 35S-Met to PUREfrex reacton solution, labelled MISTIC with a radioisotope and detected it by Photostimulated luminescence (PSL: BAS).

As we could observe the band at the aimed position (13.6kDa), we considered that MISTIC protein was expressed correctly in PURE frex.

2. Purification

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

We purified products of PUREfrex using Histag columnand by SDS-PAGE.

As we could see MISTIC in the elusion fraction and impurities were few, we concluded that the purification was succeeded.

(ⅳ) Confirmation of the introduction of unnatural amino acids

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

We would assay that that unnatural amino acids are included in MISTIC, by confirming that MISTIC expresses with suppresser tRNA and but not without the suppressor tRNA. We detected MISTIC labelled by a radioisotope by PSL (same as the method explained in “3.Expresion”).

As we could see that MISTIC is not expressed when unnatural amino acids are not added, we could determine that tRNA which sets into amber competitively with RF1 exists little. From this result, we could confirm that unnatural amino acids are included in MISTIC.

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

<img src="http://openwetware.org/images/f/ff/Izuta-5.png" width="240px">
Fig.1-1(b)-5. SDS-PAGE of PURE products

We confirmed accessibility of streptavidin(SA) to biotin under the non-denatured condition by certifying whether or not MISTIC are removed by the magnetic beads which is coupled to SA. To exclude the influence of other PURE factors, we labelled Mistic by 35S-Met and assayed by PSL (this method is mentioned above).

We loaded same amount of the product of PURE to all lanes. Lane shows that the amount of Mistic with biotin is decreased; indicating that the introduced biotins are accessible by SA.

(vi) Confirmation of Cysteine residue modification of MISTIC to cysteine

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

We labelled MISTIC by Cy5 maleimide (Cy5-MA) and confirmed whether cysteine residue of MISTIC is able to be modified or not.
We mixed 0.3µL of 10mM Cy5-MA and 50µL of 3.26µM MISTIC and incubated for 30 minutes. Products were conformed by SDS-PAGE after G-50 column purification and Cy5 fluorescence was observed. After that, protein was stained by SYPRO ORANGE and fluorescence was observed again.

As we could see the fluorescence of Cy5 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 Cy5-MA, the modification rate is presumed to be --%.

1-1(c). Design of 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) Evaluation of 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 pictures observed with both of cy3 and SYPRO-Orange, a band was observed at the same position in lane 3. Therefore, it was confirmed that Hind3 was labelled with cy3-oligonucleotide. (Multiple band of Hind3 was from the first.)

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) Evaluation of enzyme activity

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

 <img src="http://openwetware.org/images/8/86/Hind3_reactionKashiwa.png" width="280px">

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.

&8546

Fig.1-1(c)-11. Access evaluation mechanism.

(i) Evaluation of required distance between DNA origami and recognition-sequence

In this experiment, how much distance between DNA origami and recognition-sequence is required to work enzyme was evaluated. If the substrate of enzyme is too close to other structure, enzyme may not be able to access to it.

We used barrel-shaped DNA origami as an obstacle and substrate dsDNA was attached to it. Whether the enzyme can access to substrate was analyzed with Native-PAGE.

・HindⅢ Method 1) Annealed barrel structure with substrate of enzyme. 2) Reacted enzyme with barrel. 3) Analyzed by Native-PAGE 4) Observed with Cy3 5) Stained the gel by SYBR-Gold. 6) Observed by the LAS-4000

(ゲル写真　中川くんから)

Result

HindⅢ had an activity in all conditions, and when recognition-sequence was located in a position 11 bases from barrel, HindⅢ had the highest activity.

・Lambda Exonuclease It’s reaction product is ssDNA and nucleotides. So, it’s difficult to detect whether it acts to substrate and we couldn’t evaluate the access range of Lambda Exonuclease.

Since then, HindⅢ was adopted as the Activator.

1-2. Embedment of 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. Linkage of the Activator to the liposome

1-4. Control of the Wall conformation

Wall and Mistic form the complex through avidin-biotin binding using divalent SA on the membrane and biotins on each. On Mistic’s side, biotin was integrated into the polypeptide of Mistic itself using suppressor tRNA in translation. On Wall’s side, biotin is connected to Wall with dsDNA linker (22 bp) which contains restriction endonuclease recognition site.

The sequence of linker is as follows.
ssDNA1:
ATGGATGGTAAGCTTCTTCTCGtttttttTTAATATATGTGAGTGTTAATTAGGGG GAGGCGGTT
ssDNA2:
[Cy3]-CGAGAAGAAGCTTACCATCCATtttttttttttttttttttt-[Bio]

underlined part are annealed into Wall and, red letters indicate recognition site of Hind3.

Formation and decomposition of Wall- SA complex in solution

We mixed Purified Wall and SA-Alexa647 and incubated for 1h at room temperature. Then we added Hind3 and incubated for 1h again at room temperature.

The formation and decomposition was conformed by agarose electrophoresis and Cy3, Cy5 and EtBr fluorescent were observed.

In the gel images, Cy3 signal indicates the position of Wall before Hind3 treatment, but low melting temperature of the Hind3 cleaved product induce the release of Cy3 containing fragment, therefore after Hind3 treatment, the Cy3 signal indicate merely the cleaved dsDNA fragment. Also Alexa647 and EtBr signal indicates the position of SA and DNA origami, respectively.

In lane 2 new band containing both Cy3 (biotin-oligonucleotide) and Alexa647 (SA) signal was observed, indicating the formation of the Wall-SA complex.

Besides that, we conformed the Hind3 induced decomposition of Wall-SA complex by the diminishing of complex band in Cy3 and Alexa647.

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Biomod/2014/Kashiwa/Receptor

Contents

<a name="background"> 1. The Sensing System: The Receptor</a>

1-1. Preparation of the components

1-1(a). Folding of the Wall

1-1(b). Production of MISTIC

1-1(c). Design of the Activator

1-2. Embedment of the Wall into the liposome

1-3. Linkage of the Activator to the liposome

1-4. Control of the Wall conformation

Formation and decomposition of Wall- SA complex in solution

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