Biomod/2012/Tianjin/Project/YDNA

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<div id="frametext"><br><br><span style="font-size:300%;">T</span>he Y-DNA was synthesized from three ssDNAs, each of which has partial complementary sequences to the other two ssDNAs. The Y-DNA we produced only contains a blunt end, which makes it unable to polymerize. In order to polymerize, we need to create a sticky end in each branch, then use T4 ligase to link these ends.</div>
<div id="frametext"><br><br><span style="font-size:300%;">T</span>he Y-DNA was synthesized from three ssDNAs, each of which has partial complementary sequences to the other two ssDNAs. The Y-DNA we produced only contains a blunt end, which makes it unable to polymerize. In order to polymerize, we need to create a sticky end in each branch, then use T4 ligase to link these ends.</div>

Current revision

  • Background
  • The Logic Gate
    DNAzymes are DNA molecules that have the ability to perform a chemical reaction, such as catalytic action.
  • Y-DNA
    Y-DNA is composed of three ssDNA that is complementary of each other.
  • The Origami Amplifier
    DNA origami is the nanoscale folding of DNA to create arbitrary two and three dimensional shapes at the nanoscale.


The Y-DNA was synthesized from three ssDNAs, each of which has partial complementary sequences to the other two ssDNAs. The Y-DNA we produced only contains a blunt end, which makes it unable to polymerize. In order to polymerize, we need to create a sticky end in each branch, then use T4 ligase to link these ends.

Y-DNA

The Y-DNA was synthesized from three ssDNAs, each of which has partial complementary sequences to the other two ssDNAs. From last part, we know that our logic gate has a high selectivity and is a precise logic gate. It occurred to us that we can utilize this logic gate to control the formation of Y-DNA by making the logic gate one of the ssDNAs. Without adding any ions, the logic gate remain in its original structure, which leave no sequence available to form Y-DNA with other two ssDNAs. If we add Cu2+, the Cu2+ DNAzyme will self-cleave and reveal a long sequence, making it feasible to form a Y-DNA. Moreover, we knew how many logic gate have self-cleaved according to our research in the previous part, so we can control the cut logic gate generated. In this way, we can control when and how many Y-DNA be produced.

Figure 10. The design of the controlled Y-DNA formation. (From BIOMOD Team Tianjin 2012).

Because we want to use the logic gate to form Y-DNA, we need to specifically investigate its behavior in Y-DNA formation. Using NuPack (Figure 11.), we predicted that our original logic gate may fail. First, we simulated the scenario when there is only the logic gate and two ssDNAs in the solution. The result showed that the three DNA strands formed Y-DNA, because the purple region is too long, so that the two ssDNA structure can open the logic gate through strand replacement even without the activation of Cu2+. Therefore, we add a stem-loop structure by altering the sequence and adding several basepairs. In this way, Y-DNA can only form after adding Cu2+.

Figure 11. The NuPack simulation of the original design. (A): the logic gate and other ssDNA forms a Y-DNA without adding Cu2+; (B): the EcoRI digested sequence is too short to form stable duplex in one branch. (From BIOMOD Team Tianjin 2012.)
Figure 11. The NuPack simulation of the original design. (A): the logic gate and other ssDNA forms a Y-DNA without adding Cu2+; (B): the EcoRI digested sequence is too short to form stable duplex in one branch. (From BIOMOD Team Tianjin 2012.)

Subsequently, we simulated whether the improved logic gate would form a stable Y-DNA after EcoRI digestion. Again, the result didn’t give us a positive feedback. According to Figure 11., the sequence after digestion would be too short and too close to 8-17’s catalytic core to form a stable duplex. Therefore, we lengthened the sequence to ensure a stable Y-DNA after digestion. The function of this optimized new logic gate were investigated in the wet lab.

Figure 12. The NuPack simulation of the optimized design. (A): two ssDNA forms partial duplex while the logic gate molecules remains unchanged without adding Cu2+; (B): the major molecules in the solution is Y-DNA when adding Cu2+, and (C): the structure stays stable after EcoRI digestion. (From BIOMOD Team Tianjin 2012.)
Figure 12. The NuPack simulation of the optimized design. (A): two ssDNA forms partial duplex while the logic gate molecules remains unchanged without adding Cu2+; (B): the major molecules in the solution is Y-DNA when adding Cu2+, and (C): the structure stays stable after EcoRI digestion. (From BIOMOD Team Tianjin 2012.)

In conclusion, we not only repeated the conventional method of producing Y-DNA, but also introduced the logic gate to control when and how many Y-DNA are produced in the system. Next we’ll investigate how to make the Y-DNA polymerize in a controlled way.

Polymerization

The Y-DNAs we produced only contains a blunt end, which makes it unable to polymerize. In order to polymerize, we need to create a sticky end in each branch, then use T4 ligase to link these ends. In this way, the Y-DNAs are able to connect with each other, and form a large polymer. The Y-DNA is unable to form a linear structure due to its own structure. We thought that AFM can be used to determine its property.

There are two ways to have the sticky end: 1. designing it in the ssDNA from the beginning; 2. Using digestion to create the sticky end from blunt end after the formation of Y-DNA. In our experiment, we used the second way. Why? Because polymerization requires ligase to link Y-DNA together. Ordinarily, the sequences we ordered from companies contains a hydroxyl group at 5’ instead of a phosphate group that is essential for T4 ligation. While the sticky end created using enzyme digestion contains the phosphate group. Therefore, we chose to create the sticky end after the formation of Y-DNA.

EcoRI is used to digest Y-DNA, because EcoRI leaves a sticky end of AATT, which makes all sticky ends complimentary with each other. This eliminates the need to use multiple enzymes.

After we created the sticky end to every Y-DNA, we are able to polymerize all the Y-DNA by adding T4 ligase. In this way, countless Y-DNA molecules will be ligated with each other to form large polymer. If the concentration is high enough, the Y-DNA can even form a gel!

Decomposition

In the formed Y-DNA polymer, the catalytic core of 8-17 is still remain on the branch. Therefore, we can use the catalytic core to cut one of branch.

Figure 13. Decomposition of the polymer of Y-DNA with the existence of Pb2+. (From BIOMOD Team Tianjin 2012).

We know that 8-17 can only cleave substrate at a RNA site, so if the corresponding cleavage site of 8-17 on the Y-DNA is replaced with RNA, we are able to use the logic gate to control the decomposition of Y-DNA. By adding Pb2+ into the Y-DNA polymer, 8-17 is activated to cut one branch at the RNA site in every Y-DNAs. As a result, the whole polymer structure will disintegrate accordingly. As we know, there is not a effective way of control the decomposition of Y-DNA before, but our idea solved the problem. In this way, the logic gate shows its outstanding role in the decomposition of Y-DNA.