Biomod/2012/Titech/Nano-Jugglers/Methods

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:    We designed five DNA strands. DNA strands relate to "amino modified DNA and polystyrene conjugation". One DNA strand is modified amino group to conjugate polystyrene-beads. The other one is a complementary strand, which is modified thiol group to conjugate platinum. Another DNA strand also is a complementary strand, which is modified FAM. On the other hand, Two DNA strands relate "SAM".One DNA strand includes azobenzene to photo-control. Another DNA is a complementary strand, which is modified thiol group to conjugate gold.
:    We designed five DNA strands. DNA strands relate to "amino modified DNA and polystyrene conjugation". One DNA strand is modified amino group to conjugate polystyrene-beads. The other one is a complementary strand, which is modified thiol group to conjugate platinum. Another DNA strand also is a complementary strand, which is modified FAM. On the other hand, Two DNA strands relate "SAM".One DNA strand includes azobenzene to photo-control. Another DNA is a complementary strand, which is modified thiol group to conjugate gold.
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We designed these DNA strands using NUPACK*1. And we selected orthonormal sequence, referring to a paper*2. When we designed DNA strands, we fixed the temperature at 25.0 °C and the Na+ concentration at 0.195M. And we selected DNA strands, which didn't form secondary structure.  
+
:We designed these DNA strands using NUPACK*1. And we selected orthonormal sequence, referring to a paper*2. When we designed DNA strands, we fixed the temperature at 25.0 °C and the Na+ concentration at 0.195M. And we selected DNA strands, which didn't form secondary structure.  
:    1) NUPACK nucleic acid package. http://www.nupack.org/
:    1) NUPACK nucleic acid package. http://www.nupack.org/
:    2) Tetsuro Kitajima, Masahiro Takinoue, Ko-ichiroh Shohda, and Akira Suyama.
:    2) Tetsuro Kitajima, Masahiro Takinoue, Ko-ichiroh Shohda, and Akira Suyama.

Revision as of 07:19, 5 October 2012


Contents


    We have set 3 goals, that is "Energy production for rail-free movement", "Speeding up", and "Direction control".

Methods

Vapor deposition of Au and Cr on the polystyrene body

    Vapor deposition is a method for forming a thin metal film on the surface of the substrate. In this project, Gold and Chromium were deposited on the polystyrene beads in order to conjugate specific DNA onto the determined location on the surface.
>>see more Method
>>see Result
Image: BIOMODPVDM1.png

1st Deposition

First, we deposit gold on the polystyrene-bead in order to make gold hemisphered body.

2nd Deposition

Next, we change the direction of beads, and deposit chromium on the body. Then we pick up polystyrene-bead, which are deposited by quarter gold and half chromium. By chromium capping, this "head part" is prevented to conjyugate DNA strands, so platinum only mounted at the rear.

After PVD

Through twice of physical vapor deposition,
  • we conjugate thiol modified DNA onto gold part.
  • we conjugate amino modified DNA onto polystyrene part.
  • There are no DNA staple strands on chromium hemisphere.
Therefore, platinum particles can't conjugate onto chromium hemisphere.
As a result, JET starts to move toward chromium hemisphere heading.

DNA conjugation

SAM

    Self-assembled monolayers (SAM) of molecules are molecular assemblies formed spontaneously on substrate surfaces by chemical adsorption. This chemical reactions can be organized into large ordered domains and it is also possible to cover the substrate with functional end group that has some modification. In addition, few steps and experimental technique are required. SAMs are created by adsorption of head groups onto a substrate followed by a slow organization of tail groups.

    First, head groups are absorbed onto substrate over . In this phase, the surface of substrate is patterned with spots by dsorbeate molecules rather than beautiful membranes. Over the period of disordering form, the head groups assemble together on the substrate, while the tail groups assemble far from the substrate. Areas of concentrated molecules nucleate and grow until the surface of the substrate is covered in a single monolayer.

Thiol conjugation onto gold
    Thiolated compound and gold substrate are one of the most famous research of SAMs. Thiol modified DNA is conjugate to gold particles by this chemical reaction. Initially, substrate gold are cleaned by water or acid-base buffer in order to washout organic impurities. Gold particles are dissolved in 10mM phosphate buffer pH8, 10μM thiol modified DNA. Then in addition to NaCl, raise up to 0.7M salt concentration gradually over a day.Last,clean up again by proper buffer and dry. This reaction is also use ​​with platinum In order to accomplish our project ,we need platinum-DNA and gold-DNA conjugation, so we use thiol modified DNA.

>>see Result


EDAC

    We bond polystyrene-beads with DNA using EDAC. EDAC conjugation is the method to conjugate animo modified DNA onto carboxylated polystyrene surface. By using this method, we can connect platinum to aircraft body part of polystyrene regiospecifically.

    EDAC is known as water-soluble carbodiimide (WSC) and is used as coupling agent to form amide bond. EDAC crosslinks nucleic acids to the surfaces, which have carboxy group. In this project, we used this method to conjugate amino modified DNA onto carboxylated polystyrene-beads. EDAC reacts with carboxylated polystyrene-beads to form active intermediate on the body. Then it reacts to amino modified DNA. The beads are coupled by nucleic acid. We divide the body into three parts including polystyrene surface, so it is possible to connect platinum regiospecifically.
>>see Result

DNA Design

>>see more Method
    We designed five DNA strands. DNA strands relate to "amino modified DNA and polystyrene conjugation". One DNA strand is modified amino group to conjugate polystyrene-beads. The other one is a complementary strand, which is modified thiol group to conjugate platinum. Another DNA strand also is a complementary strand, which is modified FAM. On the other hand, Two DNA strands relate "SAM".One DNA strand includes azobenzene to photo-control. Another DNA is a complementary strand, which is modified thiol group to conjugate gold.
We designed these DNA strands using NUPACK*1. And we selected orthonormal sequence, referring to a paper*2. When we designed DNA strands, we fixed the temperature at 25.0 °C and the Na+ concentration at 0.195M. And we selected DNA strands, which didn't form secondary structure.
    1) NUPACK nucleic acid package. http://www.nupack.org/
    2) Tetsuro Kitajima, Masahiro Takinoue, Ko-ichiroh Shohda, and Akira Suyama.
    Design of Code Words for DNA Computers and Nanostructures with Consideration of Hybridization Kinetics. LNCS 4848, 119-129 (2008)

Observation of platinum hemisphere behavior in solution of H2O2

DNA hybridization in solution of H2O2

    H2O2 has strong corrosion. There was a risk that DNA strand is denatured by H2O2. So, we had to ensure that the hybridization of DNA is not suffer from H2O2. We guess that if DNA had been destroyed, then molecular mass would decrease in that DNA can't hybridize. Measure the relative magnitude of the molecular weight by electrophoresis, we are able to examaine whether DNA can hybridize or not.
    This time, we have to examine the state of the DNA in the time we need to steer rocket, set the time to soak and concentration of H2O2. We used PAGE electrophoresis to ascertain the stability of DNA duplex in thin H₂O₂ solution 1%~5%. PAGE electrophoresis shows the difference of molecular weight that comes from denaturetion or hybridization in the form of bands.
>>see Result

Analysis of platinum by High-speed camera

By using highspeed camera, we studied how the catalytic engine produce the driving force.In this study, we had 3 inference.
Driving force is
  • 1.produced in concurrence with the generation of bubbles
  • 2.produced as the bubbles are destructed
  • 3.produced as the bubbles detach
To research these problems, we analyzed when the moving speed of platinum beads and the acceleration of that

would change.

Energy production by using catalase

Dissociation of azobenzene-modified DNA by UV-light irradiation

>>Result page is here
    The duplex-forming activities of DNA can be photomodulated by incorporation of an azobenzene unit. The duplex is dissociated on isomerizing the trans-azobenzene to the cis form by irradiation with UV light. The duplex is formed again when the cis-azobenzene is converted to the transazobenzene by irradiation with visible light.
We inserted this function into a bead-body and add the photo-switching function to the body.In brief, so that we can detached the engines by irradiation with UV light, we attached a part of engines to the body with DNA duplex incorporating azobenzene unit.

DNA design

Image1 in detail

  • 5’ -(HS)-AATxACxCCxAGxCC-3’ (x=azobenzene)

1.We incorporated 4 azobenzene units into 11 bases DNA so that we can quickly dissociated DNA duplex.
2.5’ end of this DNA was modified by a thiol group(HS-) for forming Au-thiol bond with Au covered bead body.

Image2 in detail

  • 5’-(HS)-GGCTGGGTATT-3’

1.This 11 bases DNA are a complementary sequence of DNAⅰ(photoresponsive DNA).
2.5’ end of this DNA is modified by a thiol group(HS-) for forming Pt-thiol bond with Pt.

Ascertain the photo-switching system

  • Spectrophotometer
    We measured DNA absorbance and ascertain the duplex-forming and duplex-dissociation activities of DNAⅰ and ⅱ. As the ordered regions of stacked base pairs in the DNA duplex are dissociated, the UV absorbance increases. This difference in absorbance between the duplex and single strand state is the result of nearest neighbor base pair interactions. In other words, when the DNA is in the duplex state, interactions between base pairs decrease the UV absorbance relative to single strands. When the DNA is in the single strand state the interactions are much weaker,due to the decreased proximity, and the UV absorbance is higher than the duplex state[image3].
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