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<ul>

  	    <li class='active '><a href="http://openwetware.org/wiki/Biomod/2014/Tianjin"><span>Home</span></a></li>
  	    <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html"><span>Idea</span></a>
  	        <ul>
  	            <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#background'><span>Background</span></a></li>
                   <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#motivation'><span>Motivation</span></a></li>
                   <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#calculation'><span>Calculation</span></a></li>
 	        </ul>   
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           <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html"><span>Project</span></a>
  	        <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#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#dna origami"><span>DNA Origami</span></a></li>
               </ul> 			
           </li>   	
  	    
           <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/members.html"><span>Members and Acknowledgement</span></a>
           </li>

</ul> </div>

Strand Replacement Reaction

<html></a></html> 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

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.

Purpose

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.

Inspiration

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.

“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.”

Design

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.

Irreversible release

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.

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.

Structure

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.

Logic

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.

And the secondary reaction mechanism in fig.6 can use this kind of way to express like fig.7.

Improvement

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.

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.

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).

Synthesis of AU-DNA-CY3

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.

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.

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.

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.

@@ Line 480: / Line 480: @@
 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>]]
+[[Image:%E8%BF%9E%E7%8E%AF2.svg|thumb|550px|center|Fig 3.<br>]]
+[[Image:20140826091204.jpg|thumb|550px|center|Fig 4.<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:药物释放动画.gif|thumb|550px|center|Fig 5.<br>]]
-[[Image:药物作用示意图.png|thumb|550px|center|Fig 5.<br>]]
+[[Image:药物作用示意图.png|thumb|550px|center|Fig 6.<br>]]

Biomod/2014/experiment.html: Difference between revisions

Revision as of 20:18, 31 August 2014

Strand Replacement Reaction

Synthesis of AU-DNA-CY3

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