Bio-Nanotech. Design

Bio-Nanotechnology Design

Principal Researcher: Hao Yan

Assistant Professor: Yan Liu

Graduate Assistant: Jeanette Nangreave

MSTF Fellows: Jameel E. Hendricks & Nema Udom



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Nema, Jeanette, and Jameel

A bold new approach; Overcome the limitations of injury; Secure a safer world; Prevent and cure disease; Renew and sustain the environment; Our mission... These statements are what you see when you come into the Biodesign Institute, Arizona State University…
www.biodesign.asu.edu/research/research-centers/single-molecule-biophysics
www.biodesign.asu.edu/labs/yan/research


How did the end of our immersion at the Biodesign Institute, Arizona State University, suddenly sneak up on us? Five weeks felt like 5 hours. It is a nice place to work. A distinguished work ethic pervades (is glaringly evident) and we, two high school teachers from Arizona, were ecstatic about designing and creating, by way of self assembly, some linkage points and junctions as well as two different 2-D (2-dimensional) rectangular origami shapes in just a matter of 4 weeks. We worked withn the Hao group.

http://chemistry.asu.edu/faculty/hao_yan.asp
http://chemistry.asu.edu/faculty/Yan_Liu.asp

We appreciated the seriousness and the professional business-like attitude of everyone and their generosity in giving us so much of their precious and valuable time.
To properly identify this group, they are officially known as the SMB lab (SINGLE MOLECULE BIOPHYSICS lab). The who is the Hao group. These are housed in the beautiful new-looking state-of-the-art building called the Biodesign Institute, a building ASU is very proud of. The lead researchers are primarily from the departments of Biochemistry and Chemistry, but the research program and teaching interests of the group are highly interdisciplinary and combine Biochemistry, Chemistry, Biology, Physics and Material Science. There is a lot of great research and advancement here. The research is new and innovative, and because it is true research, outcomes are unknown and researchers don’t know if some of the things they try will work. This group is very conscientious and productive, making a great contribution to science advancement in more ways than one. Several world class journals have been published by members of this group, for example.
The common thread in this research group is that all its members work with DNA in one way or another. One ultimate goal for this particular research is to successfully achieve programmed design and assembly of biologically inspired nanomaterials. In other words, make man-made artificial nanomaterials that mimic biological nanomaterials such as DNA. The second ultimate goal is to discover new applications in biosensing, nanoelectronics, and be able to control molecules like DNA in their interactions with other things. The race is on in hundreds and thousands of research groups all over the world to be innovative, revolutionary and on the cutting edge of science in the quest for what is new, faster, stronger, better, etc….


Introduction of DNA and nanotechnology by ASU's very own Hao Yan.




Curious About Nanotechnology? Let us help you explore it





What Do We already Know?

Think about words like scotch tape, staples, Velcro, sticky ends, scaffolding, mortar, brick, wire, rope, folding, origami, shapes, design, architecture, engineering, cutting, folding, tools, self-assembly…
Think about sizes. Picture one inch (1”). An inch is about 2.5 centimeters long. Chop up a straight line that is one inch long in the lengthwise direction, into ten million parts and you will be at the nanometer level. A millimeter broken into a million parts produces pieces 1 nm long. A human hair is roughly 50,000 nm thick. www.nanotechireland.com/whatisnano.html

Consider having the ability to make, alter and manipulate something as small as 90 nm or even 15 nm or less.

Nanobiodesign and Nanotechnology are like art, science and engineering at the nano level. Tools are needed to make structures of any size, and as scientists invent these tools they are also trying to discover useful ways to use the manufactured nano materials in medicine, agriculture, security and in any way possible. For example, can we properly attach metals to DNA structures or to artificial man-made structures that mimic DNA and make circuits and nanochips?. Nanostructures hold promise in the study of cell-imaging and cell-interactions. Perhaps we can transfer different forms of energy via nanostructures.


Exploration
Making DNA structures is and can be very eye-opening. Images are very interesting to look at. Beautiful. The work itself is exciting, satisfying, rewarding. It is awesome to successfully design something so small ! Once a structure has been successfully developed and then synthesized, one must then try to tackle engineering problems.
An example of engineering is when (i) a structure is developed (ii) the structure is then synthesized.
Using DNA in engineering and architecture and art is smart because DNA is (i) a smart molecule (ii) stable (iii) predictable (iv) unique in its self-assembly properties. An example of one way to use DNA is as scaffolding to which various other things such as proteins, metals etc. are attached. In this way miniaturette nano size devices may be built.




Week one Project: Purification of DNA


Purification of DNA using the Gel Electrophoresis process
Before starting to work with any chemicals and gels, we needed a good feel of what a DNA strand looks like and how it may interlink due to its physical makeup as well as its chemical properties... At this point we were not thinking about the purely genetical properties of DNA but rather, of DNA’s tendencies to connect with something near to it. We were thinking about DNA’s self assembling properties and tendencies to stick to other things. We built two physical models of DNA from scratch using the STRAW-AND-JACK model. Then we made a connection between the two.
See Fig 4.1.


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Fig 4.1 Connection of our two single strands of DNA. The blue is disconnected from the blue, the green is disconnected from the green, and the greens are connected to the blues. The two strands are now interlocked and do not break apart when pulled.

Interpretation of Crossing Over Stands



We needed to further see this same phenomenon of connections on computer software. We practiced building double helices of single stranded DNA using TIAMAT software.

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We made connections, producing right-handed DX tiles and right-handed Holliday Junctions on the computer screen.
By Week 2, our goal is to make real connections between DNA strands, during lab practicals, using a known number of predetermined base-sequences of pure single DNA strands. We want to link or twist or fold them with each other, interlocking them together at a junction or two (Remember, up until this point, we have previously made only model-like connections with straws-and-jacks and with computer software).
Our first goal in Week 1, therefore, was to purify the DNA mix. A side ambition of ours was to learn and master the Gel Electrophoresis technique for possible use later at our high schools. Generally, the desired DNA is ordered from companies such as Integrated DNA Technologies. These companies know how to make (or mimic) DNA, otherwise known as synthetic or artificial DNA, but nevertheless authentic DNA strands. Once purchased by the research lab, the unwanted DNA strands and other impurities must be removed from the samples. Since we can’t see single DNA strands with the naked eye to pick out the good ones and separate them from the ones we didn’t want, we used a gel, a molecular sieve, in a setup called the gel electrophoresis process. The materials and methods we used are widely and routinely employed in this kind of research. This one is called the ‘Denaturing-PAGE Using the Amersham Setup (Ruby600) (For the purification of single strand synthetic DNA)’. Here is the procedure:


1. Be diligent about safety protocol – lab coat, gloves, UV-blocking Face shield, etc.
2. Check that equipment is assembled and working properly - UV transilluminator, camera, dark room, water bath, heat blocks, Eppendorf micro-pipetters, pipette tips, etc.
3. Check/prepare buffer solutions, reagents for gel preparation, DNA samples etc.
4. With de-ionized water and acetone, carefully clean the tools in which the gel will be prepared and cast, namely the Erlenmeyer flask, spacers, glass plates, combs,etc.
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5. Follow the D-PAGE Gel recipe, adding correct amounts of 20% polymix, 0% D-Page mix, 10% APS, TEMED, and either bromophenol blue dye or xylene cynole FF dye depending on the number of base pairs of the DNA strands…
6. Avoid air bubbles and leaks by keeping rubber gaskets clean and dry, by using grease, by generally being careful, by gentle tapping, and by using other tacit knowledge. Let it sit for about 30 minutes.
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7. Add deionized water to the powdered DNA samples in microtubes, mix by vortexing, then centrifuge to remove any moisture from the walls of the microtubes.
8. Add the appropriate loading dye, mix, and vortex…

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9. Cook for 5 min on a heat block at 90 degrees Celcius to denature any hydrogen bonding.
10. Continue to prepare gel assembly by taking out the comb and rinsing the wells.
11. Load the samples into the wells using the gel-loading tips. Be careful not to puncture the gel or flush the sample out of the well. Record/mark the sample sequence on the gel.
12. Load the whole gel apparatus into the electrically-run gel electrophoresis chamber and run the gel for 2-3 hours at constant ampage and at a voltage between 100V and 400V, as appropriate.MSTF_Summer_Research_mre_pics_007.jpg















13.
Turn off the power supply and circulating water.
14. Lift the gel out and put into the glass tray for EB staining, 5 min.

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15.
Transfer gel to tray of deionized water, 5 min.
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16. In the dark room, place gel on moist UVT and view. You may take a photo if desired.

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17.
Use razor blade to cut out the major bands and discard the extra gel. Further extract and elute and purify by chopping with razor, using freeze-thaw cycle, adding elution buffer, butyl and ethyl alcohols, shaking overnight in cold room…
18. Use the Spin X column filtration centrifuge tubes to separate the purified DNA from the rest of the gel.
19. Collect pure washed precipitated DNA solid in centrifuge and dry, for 2 hours in vacufuge at 300C

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20. Add 50 microliters nanopure H2O and vortex for 1 min to dissolve pure DNA fragments.
21. Measure DNA concentration in a biophotometer with printer (spectrophotometer).
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22. Carefully calculate on paper with a calculator the amount of each sample needed to make a final concentration of 30 pmol/ul.


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Fig. 4.2 Adding the dyes, Bromophenol Blue and Xylene Cylene FF.
These two chemicals help in the abstraction of different
DNA Strands in the gel poly mix.


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Fig. 4.3 Heating DNA with a heat block (and monitoring the temperature)
to break the hydrogen bonds between the DNA strands.


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Fig. 4.4 Setting up the gel casting assembly to purify and stain the DNA.
This process is called the "Gel Electrophoresis Process"




Week two Project: Annealing of Purified DNA Strands

From Week 1, we had purified and collected 5 pieces of pure aqueous DNA to make a right-handed DX connection and 4 pieces of pure aqueous DNA to make an HJ connection. It was now time to anneal them in a cold-to-hot-to-cold (R.T. - 90degreesC – 4degreesC) environment. We did this by combining the 5 pieces together in one tube and the 4 pieces together in a separate tube and heating overnight in a Polymerase Chain Reaction Thermocycler (PCR).
MSTF_Summer_Research_mre_pics_015.jpgPolymerase Chain Reaction machine (PCR)

The PCR houses the samples while they experience hot temperatures like 90 degrees down to room temperature or colder. We hoped that all 5 pieces would be present and connected in the DX tile and all 4 present and connected in the HJ. Also, different combinations of the 5 strands and the 4 strands were annealed in tiny PCR tubes as follows:- For the DX tile, Strand 1 alone, Strand 2 alone, 3 alone, 4 alone, 5 alone, 1+3, 1+3+5, 1+2+3+4, 1+2+3+4+5. For the HJ, from left to right, Lane 1, 4 alone ( 20 bases); Lane 2- 3 alone(26 bases); Lane 3, 2 alone(18 bases); Lane 4, 1 alone(18 bases); Lane 5, 1+3(44 bases); Lane 6, 1+3+4(64 bases), Lane7, 1+2+3+4(82 bases). We did not bother to put Strands 1+2 together because they should not connect to each other…
We did our second Electrophoresis Gel run. This time, instead of using denaturing gels, we ran the samples through native gels, in which the DNA strands are very stable. Instead of purifying DNA we were now analyzing it.
Analysis: We used a white light UV Transilluminator with camera and computer attached and got excellent pictures. The Holliday Junction bands are shown in Fig 4.5. Only 4 out of 7 bands remained on the gel. The gel must have run too long, thus causing the three lighter strands to run to the end and run off the gel. Lanes 1, 3 and 4 are, therefore, band-less. Where there were only 18 to 20 bases, there were no bands. We believe that the heaviest band ran the least distance, and was, therefore, the band with all 4 strands present (82 bases). It appears we had successfully created an HJ tile.


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Fig 4.5 Far right band, Lane 7, is Holliday Junction (82 bases). Lane 2
has Strand 3 (26 bases). Lanes 1, 3 & 4 are empty.



Week three and four Project: Designed and created 2D ORIGAMI




Fig 4.6 Design and Creation of 2-D Origami Structures
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We used SARSE computer software to design a dolphin. The software has the physical and chemical behavior of DNA strands programmed into it. For example, we put a hole where we thought there should be an eye. We were able to give commands about where bases should be and where they should be deleted. Once the DNA scaffolding is determined, the staples needed to hold the structure are also determined. The staples can be short pieces of DNA, such as TAGCCCTACCAGCAGA that would stick to other parts of the DNA, folding it. See the video, Visualization of DNA origami Folding, above.




Even though DNA is really three dimensional, i.e. it occupies space in 3 different directions (or axes), we do, for our purposes, refer to structures that are created by using several DNA pieces as either 2-D or 3-D structures. If we can make a rectangular shape, it is 2-D. For example, a scientist at ASU made a 2-D structure that said ASU. If we can build DNA in yet a third direction and make either a solid or hollow box, then we now have a 3-D structure. For example, a tetrahedron has been built from DNA and so has a box with a controllable lid. See http://www.nature.com/nnano/reshigh/2009/0509/full/nnano.2009.145.html
Following a 2-D origami design on paper, we assembled the DNA called M13, (6,407 bases long), DNA staples, buffer solutions, deionized water and all necessary equipment and materials. We mixed the M13 with the staples, annealed in a PCR machine, and viewed under an Atomic Force Microscope. In one sample, we had all staples needed to make a rectangle. In another sample, we omitted certain staples in order to leave a hole in the rectangle. See Fig 4.6 for our results. We were very successful in our endeavor.




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Atomic Force Microscope


Week five Project: Wrap Up/Design of 3-D Origami Structures Using caDNAno Software

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We used caDNAno computer software to continue designing 3-D origami, which we had begun working on in week 3.

http://yanlab.asu.edu/
http://yanlab.asu.edu/Research.html

The Hao group research is focused on the following four themes:
The following quote is at

chemistry.asu.edu
1) Bio-Nanotechnology: Design of novel DNA nanostructures, implementation of the designed structure in the construction of patterned DNA arrays and nanomechanical devices. Develop modular methods to achieve biomimetic molecular motors.
2) Nanoelectronics: Utilize rationally designed DNA nanostructure to template nanoelectronic components such as nanoparticles or carbon nanotubes into functional nanodevices.
3) Macromolecule Structure Elucidation: Develop methods to self-assemble 2D and 3D protein arrays for structural determination using Electron Microscopy or X-ray Crystallography.
4) Biomolecular Imaging: Investigation of protein-DNA interactions using high resolution imaging technology such as Atomic Force Microscopy and Electron Microscopy.

Major techniques in our group include: DNA/RNA/Protein manipulation (gel electrophoresis, labeling, hybridization, PCR and footprinting, cloning), electron-beam lithography, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Electron Microscopy (EM), Fluorescence Spectroscopy, UV-Vis, Circular Dichroism (CD) and chemical synthesis.


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