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             <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html"><span>Project</span></a>
             <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html"><span>Project</span></a>
           <ul>
           <ul>
               <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#strand replacement reaction"><span>Strand Replacement Reaction</span></a></li>
               <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#Three-arm locker"><span>Three-arm Locker</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#synthesis of au-dna-cy3"><span> Synthesis of AU-DNA-CY3</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#Gold nanoparticle based photosensor"><span> Gold Nanoparticle Based Photosensor</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#dna origami"><span>DNA Origami</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#dna origami"><span>DNA Origami</span></a></li>
                 </ul>
                 </ul>
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             <li class='active'><a href="http://openwetware.org/wiki/Tianjin_protocol"><span>Protocol</span></a>  
             <li class='active'><a href="http://openwetware.org/wiki/Tianjin_protocol"><span>Protocol</span></a>  
    </li>
            <li class='active'><a href="http://openwetware.org/wiki/Biomod/2014/future.html"><span>Future Work</span></a>
    </li>
    </li>
            
            
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<div id="apDiv10">
<div id="apDiv10">


<a name="strand replacement reaction"></html>
<a name="Three-arm Locker"></html>
==Strand Replacement Reaction==
==Three-arm locker==
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In molecular biology, strand displacement frequently denotes a process mediated by enzymes such as polymerases, but the reaction as defined above is guided by the biophysics of DNA and occurs independently of enzymes. Enzyme-free strand displacement and branch migration have been studied since the 1970s, but have only been applied to DNA nanotechnology within the past decade.


DNA strand displacement  
<big>DNA strand displacement</big>


Strand displacement is the process through which two strands with partial or full complementarity hybridize to each other, displacing one or more pre-hybridized strands in the process. Strand displacement can be initiated at complementary single-stranded domains (referred to as toeholds) and progresses through a branch migration process that resembles a random walk. By varying the strength (length and sequence composition) of toeholds, the rate of strand-displacement reactions can be quantitatively controlled over a factor of 10^6. More importantly, this feature allows engineering control over the kinetics of synthetic DNA devices.
Strand displacement is the process through which two strands with partial or full complementarity hybridize to each other, displacing one or more pre-hybridized strands in the process<sup>[1]</sup>. The complementary single-stranded domains (referred to as toeholds) can be the initiator of the strand displacement which progresses through a branch migration process that resembles a random walk. By varying the strength (length and sequence composition) of toeholds, the rate of strand-displacement reactions can be quantitatively controlled over a factor of 10<sup>6</sup>. More importantly, this feature allows engineering control over the kinetics of synthetic DNA devices. <br><br>


[[Image:wiki-2'_clip_image002_0001.jpg|thumb|550px|center|Fig 1.<br>]]
[[Image:Fig001.jpg|thumb|900px|center|<html><body>Figure 1-1.</body></html>Panel a shows one example of strand displacement reaction. Single-stranded DNA molecule A reacts with multi-stranded DNA complex X to release strand B and complex Y. Panel c shows that the kinetics of strand displacement can be accurately modeled and predicted from the length and sequence of the toehold domain. Copyright (2011) Nature Chemistry Dynamic Dna nanotechnology using strand displacement reactions Ref.[1]<br>]]<br>


Purpose
[[Image:Fig002.png|thumb|300px|right|<html><body>Figure 1-2.</body></html>Secondary structure of strand A, strand B, strand C and initiator I with 4 nucleotides in each letter-labelled domain.<br>]]


We use a DNA sequence to connect the DNA origami which is loaded with functional molecules, such us antitumor drugs, with the Au nanoparticles.In this case, the function of this DNA sequence is to help releasing the drugs at certain temperature and the drugs will never go back to the nanoparticles. For another, we hope that the device enables distinct drugs released under different thermal conditions in batches.
<big>Design</big>


Inspiration
To approach the goals of local release and selective release, we should design the DNA locker which is expected to intelligently control the open and close of our device.<br> <br>
According to Peng’s paper<sup>[2]</sup>, we designed a self-locking three-arm locker, which utilize strand replacement to assemble its secondary structures. However, we also optimize the three-arm structure with the irreversible characteristics of our lock, which means that it will be not re-locked once it breaks by heating.<br><br>
This locker is composed of strand A, strand B, and strand C, acting as the main body of our lock and strand I, which plays the role of initiator.<br><br><br>


We obtain our inspiration to design a DNA duplex lock device from Peng Yin’s paper Programming biomolecular self-assembly pathways that described a hairpin self-assembly using toahold and initiator.
[[Image:Fig003.gif|thumb|300px|right]]


“The hairpin motif (A in Fig. 2a) comprises three concatenated domains, a, b and c. Each domain contains a special nucleation site called a toehold, denoted at, bt and ct. Two basic reactions can be programmed using this motif, as illustrated for the example of catalytic duplex formation in Fig. 2b. First, an assembly reaction (1) occurs when a single-stranded initiator I, containing an exposed toehold at*, nucleates at the exposed toehold at of hairpin A, initiating a branch migration that opens the hairpin. Hairpin domains b and c, with newly exposed toeholds bt and ct, can then serve as assembly initiators for other suitably defined hairpins, permitting cascading (for example, in reaction (2), domain b of hairpin A assembles with domain b* of hairpin B, opening the hairpin). Second, a disassembly reaction (3) occurs when a single-stranded domain (a* of B) initiates a branch migration that displaces the initiator I from A. In this example, I catalyses the formation of duplex ANB through a prescribed reaction pathway.”
First, the locking process is''' isothermal'''. It is capable to lock at constant room temperature with a simple displacement reaction when initiator I is added. The first step will occur with the exposed toehold of hairpin A nucleating with of I and opening the hairpin. The newly exposed single strand of A will nucleating with the exposed toehold of B and open the B hairpin. After that, the same reaction will happened and lead to the open of C hairpin binding to B. Then the final step occurs in which a single-stranded domain (a* of B) initiates a branch migration that displaces the initiator I from A.<br><br>


[[Image:20140829211343.png|thumb|550px|center|Fig 2.<br>]]
When it comes to the characteristics of this structure, to get reach to our first goal, the tree-arm structure is supposed to be with high yield to form with the help of single-strand I and minimal leakage of a system containing only A, B and C.<br><br>


Design
And second, the locker should be''' irreversible'''. If we move single-strand I away and heat the locker, the double strands will melt, the locker opens, and A, B, C will return to independent hairpins. However, without I, even if the all the hairpins are annealed together, they cannot form the three-arm junction kinetically.<br><br>


There is a brilliant idea that when the system contains I, the A and B strands will assemble spontaneously, but if we part I from the system and force A and B disassembly occurring by, for example, the heat produced by gold nanoparticles, we hope to design A and B to be likely to stay singly instead of  reassembly.
Third, we design the sequences of the three-arm structure with '''diverse melting temperature''' to reach the purpose of selective release which means to open different locks and release different drugs under various circumstances, so that we can make the decision of how much dose of drugs we release and which drugs.


[[Image:melting.png|thumb|550px|center|Fig 3.<br>]]
<big>Characterization and results</big>


Irreversible release
[[Image:Fig004.png|thumb|300px|right|<html><body>Figure 1-3.</body></html>Results of repeating the three-arm branched junction experiment of Peng’s paper. Line 1 is hairpin A. Line 2 is hairpin A,B,C and I assemble at room temperature. Line 3 is the mixture of hairpin A,B and C at room temperature(leakage). Line 4 is hairpin A,B and C annealed.<br>]]


[[Image:wiki-2'_clip_image005_0000.jpg|thumb|550px|left|Fig 4.<br>]]To make the process of releasing drugs irreversible, we need to design a kind of DNA sequence which will be inactive at the single state, while active when it is complementary.
To realize those purposes of our design, we study Peng's paper and do some changes. Then modify and select the better groups step by step to get the optimum.<br><br>
First, we try to repeat the experiment of Peng’s paper (Fig 1-3), and with the consequence we discover that with its high stabilization, the length of the sequence is a little bit long and the melting temperature is higher than our expect.


We find out the hairpin structure from Peng Yin’s paper, which can exactly accord with our idea. Because of the role of the toehold in initiating strand displacement reactions, strands can be rendered effectively inactive if the toehold domain is made inaccessible by toehold sequestering. Toehold sequestering can be achieved in a number of ways, two most common of which are hybridization of the toehold to a complementary domain and isolation of the toehold in a short hairpin structure where helix formation is difficult. Programmed sequestering and subsequent exposure of toehold domains allows precise control of order and timing over the reactions and has been used in conjunctions with toehold-mediated strand displacement to construct molecular motors, polymerization reactions, catalytic reactions, and logic gates.


<big>Modification</big><br>
We modified the three-arm locker in the following 2 aspects.<br> <br>
First, in order to increase the yield of three-arm junction in the locking process and to increase the amount of independent hairpins in the unlocking process, we decided to change the number of base pairs in each domain to determine the proper lengths of the three arms. We synthesized 29 groups of various length lockers with numbers of base pairs in each domain varied from 3, 4, 5 and 6. Gel electrophoresis showed that the sequence with 4 bps per domain had the highest yield of three-arm junction in the locking process and relatively low leakage. So we reached the conclusion that 4 bps per domain is the proper length for each arm of the locker to suit our design. And we pick up the comparatively best group''' M4-11''' to do further research.<br><br>
Second, we wanted to get a group of lockers which can open in response to different melting temperature to reach the selective release, so we choose to change the length of the complementary sequence to get different melting temperature. And based on the group M4-11, we would further modify one of the domains specifically. Domain a,b and c are beyond our choice because act as toehold, the modification of them will greatly affect the formation rate of our locker. Domain x,y are also excluded because they are in strand A, and modification of them calls for using different initiator I which makes the selective released system more complicated and difficult to form. We finally chose to modify domain z which has the least effect on the whole structure.<br><br>
We synthesized 11 groups of lockers with group M4-11 and changed the number of base pairs in domain z within the range of 4,6,8,10. Finally, we selected out a high-yield gradient of lockers with different z domain of 4,8,10 bps named '''M4-11'''(Group1), '''M4-11-8-04'''(Group2), '''M4-11-10-01'''(Group3). We also heated the lockers in a temperature range from 50℃ to 65℃ and they could open successfully. As the results we show below(Fig 1-4)(Fig 1-5)<br><br>
And to get comparatively accurate melting temperature of Group1,2,3, we also heating in smaller range of temperature(Fig 1-6).<br>


[[Image:Fig005.png|thumb|700px|center|<html><body>Figure 1-4.</body></html>verification of assemble and in irreversible for three-arm structure for Group1,2,3. In each group line 1 is assemble of hairpin A,B,C and I. Line 2 is mixture of hairpin A,B and C(Leakage). Line 3 is annealing of hairpin A,B and C.<br>]]


[[Image:Fig006.png|thumb|700px|center|<html><body>Figure 1-5.</body></html>verification of assemble and in irreversible for three-arm structure for Group1,2,3. In each group line 1 is at room temperature. Line 2 is heating at 50℃. Line 2 is heating at 59.3℃ and line 4 is heating at 65℃.<br>]]


[[Image:Fig007.png|thumb|700px|center|<html><body>Figure 1-6.</body></html>verification of assemble and in irreversible for three-arm structure for Group1,2,3. In each group line 1 is at room temperature. Line 2 is heating at 60℃. Line 2 is heating at 62.5℃ and line 4 is heating at 65℃.<br>]]


Gel electrophoresis confirms:<br><br>
1.The yield of the locker is high with I added.<br> <br>
2.Without I, at constant temperature, the yield of the locker is relatively low,which means leakage is quiet low for these three groups.<br><br>
3.Without I, annealed the hairpins A, B and C, the yield of locker is also low. So when the double strands are heat and separated, it could hardly to pair again without the initiator I.<br><br>




Structure
<html><a name="Gold nanoparticle based photosensor"></html>


[[Image:wiki-2'_clip_image010.jpg|thumb|550px|left|Fig 5.<br>]]The hairpin motif like fig.5 comprises four concatenated domains, which are indicated by red, yellow, blue and green colors. And we add a hairpin on B strand to increase the stability of individual B strand by lengthening of complementation domains and prevent the extended sequence from combine with A strand without I.
==Gold Nanoparticle Based Photosensor==
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[[Image:SsDNA-Au.gif|thumb|800px|center|<html><body>Fig 2-1.</body></html> Lasers trigger the photothermal effect and the local temperature raises above the melting temperature of the DNA duplex, which allows the nonthiolated strand to dissociate into the surrounding medium while its complementary remains attached to the gold nanoparticle.<br>]]
Functionalization of thiolated DNA on gold nanospheres was carried out by following a previous published protocol with some modifications. Briefly, the disulfide bond in the thiol-modified oligonucleotides was reduced to monothiol using TCEP (10mM, 1h) in water to activate the thiolated DNA. The DNA-SH sample was added drop by drop into the gold nanoparticle solution. The solution was then incubated for 16 h at room temperature. Finally, the resulting solution was centrifuged at 1500g for 60 min and then the supernatant was decanted to remove unbound DNA-SH.<br><br>
We use gel electrophoresis to test the DNA-GNPs. The electrophoresis experiments were performed in 2 % agarose gels at 100 V,1 h. We used 5X TBE as running buffer. Gold colloids in this size range have a deep red color and can be easily visualized in the gels. The mobility of particles on the gel depends on two factors: size and charge. The bigger the size, the slower and the higher the charge, the faster particles will migrate. In the case of negatively charged Au particles, the attachment of negatively charged DNA molecules causes in first place an increase of size that can be seen as a retardation of the band of the gel. Upon attachment of DNA, the mobility of the resulting conjugates was always moderately decreased. Therefore, in agreement with previous reports, we assume throughout this manuscript that attachment of DNA to Au nanoparticles in first order increases the effective diameter of the conjugates which can be directly seen in the retardation of the band of the conjugates in gel electrophoresis experiments. The bands of DNA-Au NP complex will move slower than those of pure Au nanoparticles.<br><br>
The bands can be extracted from the gel by cutting out the agarose piece that contains the band and immersing it into 5×TBE buffer solution. After two days, the Au-DNA conjugates could be diffused out of the gel into the buffer. The extraction procedure ensures that all DNA is really attached to the Au particles, since free DNA migrates in a much faster band.<br> <br>
For the purpose of assembling the origami-GNPs complex,i.e. attaching GNPs to DNA origami as illustrated in Figure 1, we use thiol-modified oligonucleotides (short synthetic DNA sequences), which can be loaded onto the surface of GNPs to combine GNPs and DNA. GNPs are fixed onto the DNA origami by linking them to staple strands whose 5’end are modified with lipoic acid. Upon hybridization between DNA tails on DNA origami and single staple strands on GNPs, GNPs are attached to the DNA origami, resulting in the formation of origami-GNPs complex. DNA origami is used for in vivo delivery of chemotherapeutic drugs in our project. Originally, the photothermal property of GNPs was used in biologically relevant studies to destroy cancer cells, while, in our project, it has been harnessed as a means to optically elicit the release of drugs encapsulated in DNA origami.


[[Image:Gold.png.jpg|thumb|717px|center|<html><body>Figure 2-2.</body></html>solution color reflects the appropriate concentration<br>]]


In order to know gold nanoparticles of which concentration can be easily visualized in the gels, we diluted 13nm gold nanoparticle solution with water of different volumes (the volume ratios of gold nanoparticle solution and solution are 1/1,1/10,1/25,1/50,1/100,respectively). And the result showed that when the the volume ratio is at least 1/25, the diluted 13nm gold nanoparticle solution will show a clear red band in gels.




[[Image:Gold2.jpg|thumb|200px|left|<html><body>Figure 2-3.</body></html>gels result<br>]]
[[Image:Gold3.png|thumb|200px|right|<html><body>Figure 2-4.</body></html>the result of the experiment comparing the bands of 5nm and 10nm gold nanoparticles<br>]]


Then we compared the bands of 5nm and 10nm gold nanoparticles. From the figure we could see that the bands of 5nm gold nanoparticles are single while those of 13nm gold nanoparticlse are continous. This is probably because gold nanoparticles are erratic in TBE buffer while we used 5X TBE as the gel running buffer. And our 5 nm gold nanoparticles were purchased from sigma-aldrich and 13 nm gold nanoparticles were synthesized by citrate reduction of HAuCl4. Hence the stability of our 5nm gold nanoparticles may be better than 13nm gold nanoparticles.




<html><a name="dna origami"></html>
==DNA Origami==
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<big>Design</big><br><br>


We want to design a simple two-dimensional DNA origami rectangle which can be folded by our three-arm DNA locker into a roll. The dimensions of the origami plate are properly selected so the drug stays encapsulated inside it. The final dimensions are the following:


Length: 50 nm


Width: 35 nm


Thickness: 2 nm


[[Image:Jt.jpg|thumb|550px|right|<html><body>Fig 3-1.</body></html> 1% agarose electrophoresis of DNA origami with different folding processes.<br>]] 
To enable the origami to fold into a roll, the three arm locker should be combined with it. The staple strands in two corners of the origami plate are extended with the sequences of strand A, and the staple strands in the other two corners are extended with the sequences of strand C. Strand A and strand C comes from our three-arm DNA locker M4-11-6-01(Group 3) and the one with best isothermal and irreversible properties.
We used the cadnano 2 program for our rectangle shape origami design.


Logic
The scaffold used in this design is M13mp18 single strand DNA with 7249 nt, from New England Biolabs. The desired structure and dimensions are achieved with the help of two types of staples:
 
[[Image:wiki-2'_clip_image017.jpg|thumb|250px|left|Fig 6.<br>]]To assist in programming more complex reaction pathways, we abstract the motif of fig.5 as a node with four ports like fig.6. The state of each port is either accessible (open triangle/circle) or inaccessible (solid triangle/circle), depending on whether the toehold of the corresponding motif domain is exposed or sequestered.
 
[[Image:wiki-2'_clip_image019.jpg|thumb|450px|left|Fig 7.<br>]]And the secondary reaction mechanism in fig.6 can use this kind of way to express like fig.7.
 
 
 
 
 


• core: 58 staples give stability to the whole structure.


• edge: 4 staples give stability to the edges and with extending sequences of A or C.




[[Image:PCR0.png|center]]




<big>Folding and characterization</big>


The mixture of scaffold and different staples were subjected to a thermal annealing process composed of 3 steps that allowed the folding. Different initial temperatures varying from 50.0℃ to 65.0℃ in step 2 were tested. Electrophoresis showed that low initial temperatures in step 2 have relatively high origami yield.(Fig.3-1)


[[Image:AFM.jpg|thumb|550px|center|<html><body>Figure 3-2.</body></html>AFM picture of our designed origami.<br>]]
[[Image:Device1.gif|thumb|550px|center|<html><body>Figure 3-3.</body></html>schematic diagram of our device.<br>]]


 
==References==
 
[1] Zhang D Y, Seelig G. Dynamic DNA nanotechnology using strand-displacement reactions [J]. Nature chemistry, 2011, 3(2): 103-113.<br><br>
 
[2] Yin P, Choi H M T, Calvert C R, et al. Programming biomolecular self-assembly pathways[J]. Nature, 2008, 451(7176): 318-322.<br><br>  
 
[3] Sharma J, Chhabra R, Andersen C S, et al. Toward reliable gold nanoparticle patterning on self-assembled DNA nanoscaffold[J]. Journal of the American Chemical Society, 2008, 130(25): 7820-7821.<br><br>
 
[4] Ghosh P, Han G, De M, et al. Gold nanoparticles in delivery applications[J]. Advanced drug delivery reviews, 2008, 60(11): 1307-1315.<br><br>  
Improvement
[5] Lermusiaux L, Sereda A, Portier B, et al. Reversible switching of the interparticle distance in DNA-templated gold nanoparticle dimers[J]. ACS nano, 2012, 6(12): 10992-10998.<br><br>  
 
Furthermore, we develop the configuration of B strand with three hairpins to realize a stronger stability than the double-hairpin B strand. And the following electrophoretogram shows a commendable distinction that there are single A and B separating from the strip that represents the combination of A and B.
 
[[Image:project J.png|thumb|550px|center|Fig 8.    15%PAGE gel 1:L-A      2:L-B      3:AB 70℃ slow  cooling      4:AB 70℃ rapid cooling      5:AB 80℃ slow  cooling      6:AB 80℃ rapid  cooling <br>]]
 
In addition, we find an interesting phenomenon that, along with the prolong of complementary sequence in B strand when A splits from B, B strand has a high possibility to form a stable B- dyad as shown in Figure 11. We inspired by. If we optimize the A strand and B strand to enhance the probability of B’s self-assembly into B- dyad, we will have a higher success rate of irreversible melting of duplex A and B.
 
[[Image:链置换总图2.png|thumb|550px|center|Fig 9.<br>]]
 
We use Nupack to calculation free energy of individual A, B and the complementation duplex of A and B. The result demonstrates that free energy sum of individual of A and B (-52.64 kcal/mol) can be comparable to the free energy of duplex A-B (-63.64kcal/mol).
 
[[Image:20140829211426.png|thumb|550px|center|Fig 10.<br>]]
[[Image:20140829211438.png|thumb|550px|center|Fig 11.<br>]]
[[Image:20140829211451.png|thumb|550px|center|Fig 12.<br>]]
 
<html><a name="synthesis of au-dna-cy3"></html>
 
==Synthesis of AU-DNA-CY3==
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<html><a name="dna origami"></html>
 
[[Image:DNA-Au-2.png|thumb|800px|center|Fig 1. Lasers trigger the photothermal effect and the local temperature raises above the melting temperature of the DNA duplex, which allows the nonthiolated strand to dissociate into the surrounding medium while its complementary remains attached to the gold nanoparticle.
<br>
]]
For the purpose of assembling the origami-GNPs complex,i.e. attaching GNPs to DNA origami as illustrated in Figure 1, we use thiol-modified oligonucleotides (short synthetic DNA sequences), which can be loaded onto the surface of GNPs to combine GNPs and DNA. And we choose Cy3 fluorescent dye to target the DNA strand for the detection of combination. GNPs are fixed onto the DNA origami by linking them to staple strands whose 5’end are modified with lipoic acid. Upon hybridization between DNA tails on DNA origami and single staple strands on GNPs, GNPs are attached to the DNA origami, resulting in the formation of origami-GNPs complex. DNA origami is used for in vivo delivery of chemotherapeutic drugs in our project. Originally, the photothermal property of GNPs was used in biologically relevant studies to destroy cancer cells, while, in our project, it has been harnessed as a means to optically elicit the release of drugs encapsulated in DNA origami.
 
==DNA Origami==
<html></a></html>
We use caDNAno, an open-source software for designing DNA sequences to fold 3D honeycomb-pleated shapes that were designed, assembled, and analyzed to identify a well-behaved motif, to project our DNA origami. We created a DNA nanostructure in the form of a hexagonal barrel with dimensions of 35 nm × 35 nm× 45 nm. Our DNA origami is folded from one single-stranded M13 ssDNA (∼7200 nt long) using single-stranded staple strands. Particular margins of the barrel consist of special staple strands, so that we can introduce specific secondary structure into the staple strands to achieve the locking and opening of the barrel. The barrel consists of two domains that are covalently attached in the rear by single-stranded scaffold hinges, and can be noncovalently fastened in the front by staples modified with DNA light–based locks. A clasp system based on strands displacement was generally used to control the opening of the lid on a DNA box.
 
[[Image:20140829210305.jpg|thumb|550px|center|Fig 1.<br>]]
 
[[Image:六棱柱origami打开.gif|thumb|550px|center|Fig 2.<br>]]
 
In order to operate our device in response to NIR laser, we designed a DNA light–based lock mechanism that opens in response to light excitation. AuNPs are attached to DNA origami using DNA linkers (DNA locks). After optical excitation, the AuNPs will locally generate heat to raise the temperature over the melting temperature, so that the heat breaks the DNA double strands and the nano-spaceship undergoes a drastic reconfiguration to expose its previously sequestered surfaces.
 
[[Image:20140826091204.jpg|thumb|550px|center|Fig 3.<br>]]
 
Furthermore, we simulated a new form of DNA origami which looks like the gate of our university, shown in the Figure 1, and this gate is assembled by the main part and two gate posts, all made by DNA origami. The three components are connected by the strands lock above-mentioned and attached with gold nanoparticles. And we modify the functional biomolecules beneath the main part of the gate. The gate posts jacks a space between the functional biomolecules and the receptors on the surface of cells. Only if, the open-lock reaction happens, the gate posts will separate from the main part and the receptors can have the chance to incorporate the functional biomolecules.
 
[[Image:药物释放动画.gif|thumb|550px|center|Fig 4.<br>]]
 
[[Image:药物作用示意图.png|thumb|550px|center|Fig 5.<br>]]
 




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<a name="Three-arm Locker"></html>

Three-arm locker

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DNA strand displacement

Strand displacement is the process through which two strands with partial or full complementarity hybridize to each other, displacing one or more pre-hybridized strands in the process[1]. The complementary single-stranded domains (referred to as toeholds) can be the initiator of the strand displacement which progresses through a branch migration process that resembles a random walk. By varying the strength (length and sequence composition) of toeholds, the rate of strand-displacement reactions can be quantitatively controlled over a factor of 106. More importantly, this feature allows engineering control over the kinetics of synthetic DNA devices.

<html><body>Figure 1-1.</body></html>Panel a shows one example of strand displacement reaction. Single-stranded DNA molecule A reacts with multi-stranded DNA complex X to release strand B and complex Y. Panel c shows that the kinetics of strand displacement can be accurately modeled and predicted from the length and sequence of the toehold domain. Copyright (2011) Nature Chemistry Dynamic Dna nanotechnology using strand displacement reactions Ref.[1]


<html><body>Figure 1-2.</body></html>Secondary structure of strand A, strand B, strand C and initiator I with 4 nucleotides in each letter-labelled domain.

Design

To approach the goals of local release and selective release, we should design the DNA locker which is expected to intelligently control the open and close of our device.

According to Peng’s paper[2], we designed a self-locking three-arm locker, which utilize strand replacement to assemble its secondary structures. However, we also optimize the three-arm structure with the irreversible characteristics of our lock, which means that it will be not re-locked once it breaks by heating.

This locker is composed of strand A, strand B, and strand C, acting as the main body of our lock and strand I, which plays the role of initiator.


First, the locking process is isothermal. It is capable to lock at constant room temperature with a simple displacement reaction when initiator I is added. The first step will occur with the exposed toehold of hairpin A nucleating with of I and opening the hairpin. The newly exposed single strand of A will nucleating with the exposed toehold of B and open the B hairpin. After that, the same reaction will happened and lead to the open of C hairpin binding to B. Then the final step occurs in which a single-stranded domain (a* of B) initiates a branch migration that displaces the initiator I from A.

When it comes to the characteristics of this structure, to get reach to our first goal, the tree-arm structure is supposed to be with high yield to form with the help of single-strand I and minimal leakage of a system containing only A, B and C.

And second, the locker should be irreversible. If we move single-strand I away and heat the locker, the double strands will melt, the locker opens, and A, B, C will return to independent hairpins. However, without I, even if the all the hairpins are annealed together, they cannot form the three-arm junction kinetically.

Third, we design the sequences of the three-arm structure with diverse melting temperature to reach the purpose of selective release which means to open different locks and release different drugs under various circumstances, so that we can make the decision of how much dose of drugs we release and which drugs.

Characterization and results

<html><body>Figure 1-3.</body></html>Results of repeating the three-arm branched junction experiment of Peng’s paper. Line 1 is hairpin A. Line 2 is hairpin A,B,C and I assemble at room temperature. Line 3 is the mixture of hairpin A,B and C at room temperature(leakage). Line 4 is hairpin A,B and C annealed.

To realize those purposes of our design, we study Peng's paper and do some changes. Then modify and select the better groups step by step to get the optimum.

First, we try to repeat the experiment of Peng’s paper (Fig 1-3), and with the consequence we discover that with its high stabilization, the length of the sequence is a little bit long and the melting temperature is higher than our expect.


Modification
We modified the three-arm locker in the following 2 aspects.

First, in order to increase the yield of three-arm junction in the locking process and to increase the amount of independent hairpins in the unlocking process, we decided to change the number of base pairs in each domain to determine the proper lengths of the three arms. We synthesized 29 groups of various length lockers with numbers of base pairs in each domain varied from 3, 4, 5 and 6. Gel electrophoresis showed that the sequence with 4 bps per domain had the highest yield of three-arm junction in the locking process and relatively low leakage. So we reached the conclusion that 4 bps per domain is the proper length for each arm of the locker to suit our design. And we pick up the comparatively best group M4-11 to do further research.

Second, we wanted to get a group of lockers which can open in response to different melting temperature to reach the selective release, so we choose to change the length of the complementary sequence to get different melting temperature. And based on the group M4-11, we would further modify one of the domains specifically. Domain a,b and c are beyond our choice because act as toehold, the modification of them will greatly affect the formation rate of our locker. Domain x,y are also excluded because they are in strand A, and modification of them calls for using different initiator I which makes the selective released system more complicated and difficult to form. We finally chose to modify domain z which has the least effect on the whole structure.

We synthesized 11 groups of lockers with group M4-11 and changed the number of base pairs in domain z within the range of 4,6,8,10. Finally, we selected out a high-yield gradient of lockers with different z domain of 4,8,10 bps named M4-11(Group1), M4-11-8-04(Group2), M4-11-10-01(Group3). We also heated the lockers in a temperature range from 50℃ to 65℃ and they could open successfully. As the results we show below(Fig 1-4)(Fig 1-5)

And to get comparatively accurate melting temperature of Group1,2,3, we also heating in smaller range of temperature(Fig 1-6).

<html><body>Figure 1-4.</body></html>verification of assemble and in irreversible for three-arm structure for Group1,2,3. In each group line 1 is assemble of hairpin A,B,C and I. Line 2 is mixture of hairpin A,B and C(Leakage). Line 3 is annealing of hairpin A,B and C.
<html><body>Figure 1-5.</body></html>verification of assemble and in irreversible for three-arm structure for Group1,2,3. In each group line 1 is at room temperature. Line 2 is heating at 50℃. Line 2 is heating at 59.3℃ and line 4 is heating at 65℃.
<html><body>Figure 1-6.</body></html>verification of assemble and in irreversible for three-arm structure for Group1,2,3. In each group line 1 is at room temperature. Line 2 is heating at 60℃. Line 2 is heating at 62.5℃ and line 4 is heating at 65℃.

Gel electrophoresis confirms:

1.The yield of the locker is high with I added.

2.Without I, at constant temperature, the yield of the locker is relatively low,which means leakage is quiet low for these three groups.

3.Without I, annealed the hairpins A, B and C, the yield of locker is also low. So when the double strands are heat and separated, it could hardly to pair again without the initiator I.


<html><a name="Gold nanoparticle based photosensor"></html>

Gold Nanoparticle Based Photosensor

<html></a></html>

<html><body>Fig 2-1.</body></html> Lasers trigger the photothermal effect and the local temperature raises above the melting temperature of the DNA duplex, which allows the nonthiolated strand to dissociate into the surrounding medium while its complementary remains attached to the gold nanoparticle.

Functionalization of thiolated DNA on gold nanospheres was carried out by following a previous published protocol with some modifications. Briefly, the disulfide bond in the thiol-modified oligonucleotides was reduced to monothiol using TCEP (10mM, 1h) in water to activate the thiolated DNA. The DNA-SH sample was added drop by drop into the gold nanoparticle solution. The solution was then incubated for 16 h at room temperature. Finally, the resulting solution was centrifuged at 1500g for 60 min and then the supernatant was decanted to remove unbound DNA-SH.

We use gel electrophoresis to test the DNA-GNPs. The electrophoresis experiments were performed in 2 % agarose gels at 100 V,1 h. We used 5X TBE as running buffer. Gold colloids in this size range have a deep red color and can be easily visualized in the gels. The mobility of particles on the gel depends on two factors: size and charge. The bigger the size, the slower and the higher the charge, the faster particles will migrate. In the case of negatively charged Au particles, the attachment of negatively charged DNA molecules causes in first place an increase of size that can be seen as a retardation of the band of the gel. Upon attachment of DNA, the mobility of the resulting conjugates was always moderately decreased. Therefore, in agreement with previous reports, we assume throughout this manuscript that attachment of DNA to Au nanoparticles in first order increases the effective diameter of the conjugates which can be directly seen in the retardation of the band of the conjugates in gel electrophoresis experiments. The bands of DNA-Au NP complex will move slower than those of pure Au nanoparticles.

The bands can be extracted from the gel by cutting out the agarose piece that contains the band and immersing it into 5×TBE buffer solution. After two days, the Au-DNA conjugates could be diffused out of the gel into the buffer. The extraction procedure ensures that all DNA is really attached to the Au particles, since free DNA migrates in a much faster band.

For the purpose of assembling the origami-GNPs complex,i.e. attaching GNPs to DNA origami as illustrated in Figure 1, we use thiol-modified oligonucleotides (short synthetic DNA sequences), which can be loaded onto the surface of GNPs to combine GNPs and DNA. GNPs are fixed onto the DNA origami by linking them to staple strands whose 5’end are modified with lipoic acid. Upon hybridization between DNA tails on DNA origami and single staple strands on GNPs, GNPs are attached to the DNA origami, resulting in the formation of origami-GNPs complex. DNA origami is used for in vivo delivery of chemotherapeutic drugs in our project. Originally, the photothermal property of GNPs was used in biologically relevant studies to destroy cancer cells, while, in our project, it has been harnessed as a means to optically elicit the release of drugs encapsulated in DNA origami.

<html><body>Figure 2-2.</body></html>solution color reflects the appropriate concentration

In order to know gold nanoparticles of which concentration can be easily visualized in the gels, we diluted 13nm gold nanoparticle solution with water of different volumes (the volume ratios of gold nanoparticle solution and solution are 1/1,1/10,1/25,1/50,1/100,respectively). And the result showed that when the the volume ratio is at least 1/25, the diluted 13nm gold nanoparticle solution will show a clear red band in gels.


<html><body>Figure 2-3.</body></html>gels result
<html><body>Figure 2-4.</body></html>the result of the experiment comparing the bands of 5nm and 10nm gold nanoparticles

Then we compared the bands of 5nm and 10nm gold nanoparticles. From the figure we could see that the bands of 5nm gold nanoparticles are single while those of 13nm gold nanoparticlse are continous. This is probably because gold nanoparticles are erratic in TBE buffer while we used 5X TBE as the gel running buffer. And our 5 nm gold nanoparticles were purchased from sigma-aldrich and 13 nm gold nanoparticles were synthesized by citrate reduction of HAuCl4. Hence the stability of our 5nm gold nanoparticles may be better than 13nm gold nanoparticles.


<html><a name="dna origami"></html>

DNA Origami

<html></a></html> Design

We want to design a simple two-dimensional DNA origami rectangle which can be folded by our three-arm DNA locker into a roll. The dimensions of the origami plate are properly selected so the drug stays encapsulated inside it. The final dimensions are the following:

Length: 50 nm

Width: 35 nm

Thickness: 2 nm

<html><body>Fig 3-1.</body></html> 1% agarose electrophoresis of DNA origami with different folding processes.

To enable the origami to fold into a roll, the three arm locker should be combined with it. The staple strands in two corners of the origami plate are extended with the sequences of strand A, and the staple strands in the other two corners are extended with the sequences of strand C. Strand A and strand C comes from our three-arm DNA locker M4-11-6-01(Group 3) and the one with best isothermal and irreversible properties. We used the cadnano 2 program for our rectangle shape origami design.

The scaffold used in this design is M13mp18 single strand DNA with 7249 nt, from New England Biolabs. The desired structure and dimensions are achieved with the help of two types of staples:

• core: 58 staples give stability to the whole structure.

• edge: 4 staples give stability to the edges and with extending sequences of A or C.



Folding and characterization

The mixture of scaffold and different staples were subjected to a thermal annealing process composed of 3 steps that allowed the folding. Different initial temperatures varying from 50.0℃ to 65.0℃ in step 2 were tested. Electrophoresis showed that low initial temperatures in step 2 have relatively high origami yield.(Fig.3-1)

<html><body>Figure 3-2.</body></html>AFM picture of our designed origami.
<html><body>Figure 3-3.</body></html>schematic diagram of our device.

References

[1] Zhang D Y, Seelig G. Dynamic DNA nanotechnology using strand-displacement reactions [J]. Nature chemistry, 2011, 3(2): 103-113.

[2] Yin P, Choi H M T, Calvert C R, et al. Programming biomolecular self-assembly pathways[J]. Nature, 2008, 451(7176): 318-322.

[3] Sharma J, Chhabra R, Andersen C S, et al. Toward reliable gold nanoparticle patterning on self-assembled DNA nanoscaffold[J]. Journal of the American Chemical Society, 2008, 130(25): 7820-7821.

[4] Ghosh P, Han G, De M, et al. Gold nanoparticles in delivery applications[J]. Advanced drug delivery reviews, 2008, 60(11): 1307-1315.

[5] Lermusiaux L, Sereda A, Portier B, et al. Reversible switching of the interparticle distance in DNA-templated gold nanoparticle dimers[J]. ACS nano, 2012, 6(12): 10992-10998.



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