Biomod/2013/Tianjin/Experiments & Results

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Polymerizing

optimize the 1st stem-loop structure & termination

Cleavage

optimize the 2nd stem-loop structure

Delivery device


Is the polymer really could exist? This is the most important question for this design. In this part, we characterize the polymerization reaction in different conditions. What’s more, we want to design a terminator for this polymer.


optimize the 1st stem-loop structure

The ability of polymerize is totally decided by the 1st stem-loop contracture, if it is too long, the specificity of the polymerization reaction will be good, which means there will be no adverse reactions, but the whole process will be very long and hard to take place. Well, if the stem domain is too short, it won’t need any inducer to go on its polymerization reaction. And to make the walker more easily contact to the DNA polymer track, we’d better keep the distance between to free tail about 10.5 or 21bp.The salt concentration will affect the efficiency of DNA binding, so we just use the origin reaction buffer as the article mentioned, HEPES 200mmol/L, NaCl 0.5mol/L.

At first, we set the stem domain as 7 base pairs. And the result turns to be disappointed. As showing in Figure3.1.1, lane 1 to 3 and 5 contains the same track monomer concentration, 2.5nmol/L. And the trigger DNA keep raising from lane1 to 3.The lane 4 is a marker, and the lane 5 is trigger free. The track monomer should be around 40 base pairs, but we can see that all the lanes have a band around 200.This shows that our DNA polymer is formed without any inducement. Because the 4 kinds of DNA track monomers have the same concentration, so the polymer should be around 200.

Figure3.1.1 1% agarose gel. Lane1, 2, 3 and 5 each contain 2.5nmol/L track monomerand the trigger is 0.5, 1.0, 2.5, 0 nmol/L. Lane four is the marker.
Figure3.1.1 1% agarose gel. Lane1, 2, 3 and 5 each contain 2.5nmol/L track monomerand the trigger is 0.5, 1.0, 2.5, 0 nmol/L. Lane four is the marker.

So we make the stem longer to 17bp, and the result also turns to be disappointed. The agarose gel did not show any sign of polymerize, as the Figure 3.1.2. So we ran another PAGE, to see if there is any other sign. As the Figure3.1.3 shows that there are some polymer exist, but just a little. It means that our design works but it still need optimization.

Figure3.1.2 1% agarose gel. Lane 4 is a maker lane 1 to 3 has the same monmer concentration as 2.5mol/L. Lane 3 and 4 have a concentration of 1, 2.5mol/L.
Figure3.1.2 1% agarose gel. Lane 4 is a maker lane 1 to 3 has the same monmer concentration as 2.5mol/L. Lane 3 and 4 have a concentration of 1, 2.5mol/L.
Figure3.1.3 10% page All the lanes have the same monomer concentration as 2.5nmol/L the trigger concentration from lane 1 to lane 6 are 0.2, 1.0, 1.5, 2.0, 2.5 nmol/L.
Figure3.1.3 10% page All the lanes have the same monomer concentration as 2.5nmol/L the trigger concentration from lane 1 to lane 6 are 0.2, 1.0, 1.5, 2.0, 2.5 nmol/L.

So at last, we decide to set the stem domain as 15bp and the distance between two free tail 21bp, which makes the track to be flat to promise a better conductivity with the walker. And the ends turns out to be excited.

We ran a PAGE as Figure3.1.4, and find that the lane with the trigger has only few monomers left and the band of the lane without trigger DNA seems much brighter. We can also see that there are some unexpected polymer, which means that there will be some adverse reaction have taken place. We also run a agarose gel as Figure 3.1.5, and find that the result was totally as our expection. The polymer remains about 400 base pairs, while the lane without trigger just has some slight adverse reaction less than 200bp. The reason for the 400bp band is that we add the same concentration of the trigger and the 4 monomers.

Figure3.1.4 Lane 1 is the sample that we added 2.5 nmol/L trigger, the second lane is just T1.lane3 is the sample we add no trigger but T1 to T4. All the lanes have a concentration of the monomer as 2.5 nmol/L.
Figure3.1.4 Lane 1 is the sample that we added 2.5 nmol/L trigger, the second lane is just T1.lane3 is the sample we add no trigger but T1 to T4. All the lanes have a concentration of the monomer as 2.5 nmol/L.
Figure3.1.5 Lane1 is the sample that we added 2.5 nmol/L trigger, the second lane is just a maker. Lane 3 is the sample we add no trigger but T1 to T4. All the lanes have a concentration of the monomer as 2.5 nmol/L.
Figure3.1.5 Lane1 is the sample that we added 2.5 nmol/L trigger, the second lane is just a maker. Lane 3 is the sample we add no trigger but T1 to T4. All the lanes have a concentration of the monomer as 2.5 nmol/L.

To see how the reaction taken place in different temperature, we do the reaction in different and run a agarose gel. We did it in 4℃, 25℃ and 35℃, when temperature is low the reation goes very slowly, so does the adverse reaction. But as the temperature, the band keeps going us and we can see that the reaction goes faster. To saving time, all the polymers we make is under 35℃, through it will bring a little adverse reaction in absence of that trigger DNA, but it works well when adding the trigger DNA, and we didn’t see much adverse-reaction’s came out band in the lane adding the trigger.

Figure3.1.6 The 1st lane is a maker. Every 3 lane is a group, the 3 groups each take its reaction under 4℃, 25℃ and 35℃. The 3 lanes in each group mass no trigger one, high trigger concentration one and low trigger concentration one.
Figure3.1.6 The 1st lane is a maker. Every 3 lane is a group, the 3 groups each take its reaction under 4℃, 25℃ and 35℃. The 3 lanes in each group mass no trigger one, high trigger concentration one and low trigger concentration one.

But it’s not enough to prove that the polymerization reaction can be triggered, we need it to be longer and see how long it could be. So we add the concentration of the 4 monomers. And we find that the polymer have a much larger molecular weight, at about 2000bp.As, Figure 3.1.7.

Figure3.1.7 1% agarose The momomer con centration=25nmol/L the other condition is the same to the Figure 3.1.6.
Figure3.1.7 1% agarose The momomer con centration=25nmol/L the other condition is the same to the Figure 3.1.6.

Terminator

We also design a terminator for the polymer, it could bind to the free domain of T4 which could open the 1st stem-loop structure of T1.We adding the terminate DNA in different duration of the reaction and got this figure. It shows that the terminator could stop the polymerization reaction.

Figure3.1.8 Every lane has the same composition: T1 to T4 2.5nmol/L, trigger DNA 2.5nmol/L. Terminnator 2.5nmol/l. We add termianator at 2h, 1.5h, 0.5h, 0h after reaction.
Figure3.1.8 Every lane has the same composition: T1 to T4 2.5nmol/L, trigger DNA 2.5nmol/L. Terminnator 2.5nmol/l. We add termianator at 2h, 1.5h, 0.5h, 0h after reaction.



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