Biomod/2012/Titech/Nano-Jugglers/Methods

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(3-1.Design of photoresponsive DNA)
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==<font color="orange">3-1.Design of photoresponsive DNA</font>==
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==3-1.Design of photoresponsive DNA==
::<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/a/ac/Azobenzene_modified_DNA.jpg" border=0 width=300 height=240></a></td></body></html>
::<html><body><td align="center" width="150px"><img src="http://openwetware.org/images/a/ac/Azobenzene_modified_DNA.jpg" border=0 width=300 height=240></a></td></body></html>
:&nbsp;&nbsp;&nbsp;&nbsp;We incorporated 4 azobenzene units into photoresponsive DNA strand so that we can quickly dissociated DNA duplex.{Photo-switchable DNA 5'-AATXACXCCXAGXCC-3' (X=Azobenzene)}
:&nbsp;&nbsp;&nbsp;&nbsp;We incorporated 4 azobenzene units into photoresponsive DNA strand so that we can quickly dissociated DNA duplex.{Photo-switchable DNA 5'-AATXACXCCXAGXCC-3' (X=Azobenzene)}

Revision as of 19:16, 22 October 2012


0.Construction of Biomolecular Rocket

Selective coating by vapor deposition

    In order to have a positional specificity, We make metal-deposited beads. Hemispherical part of polystyrene bead is covered with Cr,1/4 part of spere is covered with Au and the other part is polystyrene. First, deposit Au on the half of polystyrene beads. Second, deposit Cr on the half of polystyrene beads.

DNA conjugation to polystyrene surface

    Carboxyl (COOH) microparticles can be used for covalent coupling of proteins by activating the carboxyl group with water-soluble carbodiimide. The carbodiimide reacts with the carboxyl group to create an active ester that is reactive toward primary amines on the DNA.
First, carboxyl group of polystyrene beads and EDAC react with each other. Next, Carboxyl group become active intermediate. Then DNA coupled to Polystyrene beads

DNA conjugation to metal surface

    When we mix thiol-modified DNA and Pt or Au, they will be self assembly. Thiol-modified DNA and Au or Pt are organized naturally. Using this characteristic, bond two kinds of DNA with Pt and bond another kind of DNA with Au.

Catalyst conjugation

     Finally, we conjugated the catalyst to our rocket body by DNA hybridization. Complementary strand DNA had adhered to the corresponding parts of the rocket body, so it enabled to conjugate catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.     we build our rocket in this way.


0-1.Selective coating of the body

    1st Deposition     We deposit gold on the polystyrene-bead 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.

    Therefore, platinum particles can't conjugate onto chromium hemisphere. As a result, our rocket starts to move toward chromium hemisphere heading.

>back to Result

0-2.DNA design

    We use DNA strands for bonding between a body of Biomolecular Rocket and platinum particles. DNA strands must be designed 4 DNA strands to hybridize at room temperature stably. DNA strands are categorize into two types, one type is a DNA strand for conjugation to polystyrene surface and its cDNA, and another type is a DNA strand for conjugation to gold surface and its cDNA. Each cDNA conjugated to platinum particles.
    In this section, we explain how we design DNA strands. There are three steps to design DNA strands, first step is selection of DNA strands , second step is adjustment of the number of bases and final step is design of cDNA strand. We use NUPACK*1 for these processes of DNA design. NUPACK is a software for the analysis and design of nucleic acid systems.

    First, we chose DNA strands from a treatise*2. In this step, we analyze whether DNA can easily hybridize at room temperature stably. Then we select two DNA strands. Secondly, we adjust number of bases in order to make easily dissociate when they introduced photo-switching systems. Finally, we design c of each cDNA strands, and modify amino group and thiol group as necessary.
    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)
>>back to Result

0-3.DNA conjugation

0-3-1.Selective conjugation of DNA to polystyrene surface area


    We bond polystyrene-beads with DNA using EDAC. 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 each DNA with position classification. By using this method, we can connect platinum to aircraft body part of polystyrene in a region-specific manner.

>>back to Result

0-3-2.Selective conjugation of DNA to metal surface area


    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.

SAM 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, intended to raise the concentration of NaCl every 2 hours after 24 hours of incubation for 6 hours, then incubate more 16 hoursraise up to 0.7M salt concentration (Salt aging process).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.

>>back to Result

0-4.Catalyst conjugation by DNA hybridization

    We conjugated the catalyst to our rocket body by DNA hybridization. cDNA had adhered to the corresponding parts of the rocket body, so it enabled to conjugate catalyst engines in that we designed these DNA were thermodynamically stable when they hybridized.

    First, we prepared DNA-conjugated 10 μm sized polystyrene beads which were deposited 1/4 Au and 1/2 Cr. At the same time we prepared DNA-conjugated 0.15~0.40 μm sized platinum particles. Then, mixed these materials in 3×SSC buffer at 90℃, and cooled to room temperature slowly over time. Namely, annealing DNA by raising temperature at the first time, next make it easy to hybridize each other by downing to room temperature.

>>back to Result




1-1.DNA hybridization in solution of H2</subl>O2

    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.
>>back to Result

1-2.Verification of platinum hemisphere behavior in solution of H2O2

    The driving force of Biomolecular Rocket is catalytic engine of platinum. This catalytic engine become more powerful, increasing the surface of platinum. Biomolecular Rocket has numerous platinum particles to increase the surface of platinum. In this experiment, we deposit chromium to 1µm beads which are covered platinum and we make half platinum beads. In addition, we observe a behavior of the beads in 3% H2O2 solution. From this experiment, we are able to investigate whether Surface area of platinum is related to the speed.
>>back to Result

1-3.Verification of catalase hemisphere behavior in solution of H2O2

    For speeding-up of Biomolecular Rocket, we experiment with another catalytic engine. Catalase is known as a enzyme that catalyzes the hydrogen peroxide solution. And we expect that this enzyme will be new catalytic engine of the Biomolecular Rocket because a enzyme activity of this enzyme is 100 000 times stronger than that of platinum. In this experiment, we deposit chromium for half surface of polystyrene beads and catalase conjugated onto polystyrene side. In addition, we observe a behavior of the beads in diluted H2O2 solution.

>>back to Result




2-1.Analyses of the speed of platinum in solution of H2O2 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 by using "Highspeed microscope VW-9000".

>>back to Result




3-1.Design of photoresponsive DNA

    We incorporated 4 azobenzene units into photoresponsive DNA strand so that we can quickly dissociated DNA duplex.{Photo-switchable DNA 5'-AATXACXCCXAGXCC-3' (X=Azobenzene)}
    Abs
    A relational expression about absorbance can be expressed as follows.
    (Abs: absorbance, ε: the absorption coefficient, c: strength, d: length of a visual leg )
>>back to Result

3-2.Dissociation of photoresponsive DNA by UV-light irradiation

    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].
>>back to Result
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