1. Formation of Z-B DNA chimera.
Figure 1 a is the Native PAGE pattern of Z-B chimera and figure 1 b is the denaturing PAGE pattern. The samples were loaded in the same volume on the 8% Native gels and denaturing gels for characterization of Z-DNA and B-DNA. After optimizing the reaction methods (please read in the “Discussion”), the formation of Z-B DNA chimera was obtained when two complementary circular DNA were added simultaneously (shown in Materials and Methods).
We can found that the circular DNAs have formed in lane 3 and lane 4 and Z-B chimera has also formed in lane 5 when we mixed the product in lane 3 and lane 4 in the same proportion under the condition of 10mM MgCl2 in the gel and 1mM MgCl2 in the 1×TBE buffer, that means, even 42nt can form the Z-B DNA chimera. We originally assumed that no other byproduct could be seen if pairing efficiency was high enough. However, as shown in Figure 1a, in the lane 6 and lane 7, there was another band respectively, the mobility of which was slower than the band of circular DNA and it’s complementary linear DNA, we think the structure of the product is as figure 1 c.
However, when we used denaturing PAGE gel (Figure 1 b), the double strand circle DNA was not observed, indicating that the double strand circle DNA dissociated to ssDNA cicular DNAs under 8M urea as the denaturing agent (lane 5). The similar phenomenon was observed for lane 6,7,8.
The result of another length was as shown in figure 2. Figure 2 a is the Native PAGE pattern of Z-B chimera and figure 2 b is the denaturing PAGE pattern. The condition of the gel was not same as 42nt, we did not add MgCl2 either in the gel or in the 1×TBE buffer, we just add ice to keep the temperature under 20℃and samples were loaded in the same volume on the 8% Native gels and denaturing gels for characterization of Z-DNA and B-DNA. The result of gel illustration of 72nt samples were similar as 42nt in figure 1. The formation of Z-B DNA chimera was also obtained. The difference between them was as follow: the samples in lane 7 and lane 9 were duplex by single strand DNA with it’s complementary linear DNA and ligated by T4 DNA ligase to form a closed ds-DNA circle, we can find that they have the similar mobility with ds-cirDNA on native PAGE gel, and the mobility still remained low even under denaturing conditions, indicating that the closed circles can not dissociate to free circles. This experience further evident that the Z-B DNA chimera was formed by base pairing of single strand circle DNA and it’s complementary ss-cirDNA. The reason why we did not load the sample of 42nt as lane 7 and lane 9 of 72nt is that, limited to the diameter of the ring after circling of ssDNA and the of ssDNA itself, they will not form the structure we expect as figure 2 c when ss-cirDNA combined with it’s complementary ssDNA, instead, they may form the structure as figure 2 d and the closed circles as the samples of lane 7 and lane 9 of 72nt will not form.
The experiment results of other lengths were similar as 42nt and 74nt (data not shown).
Image of combining with ZBP-1:
The conditions of electrophoresis are the same as those when we proved to form the Z-B DNA chimera. We can find that the mobility of the Z-B chimera band with protein ZBP-1 in lane 6 was much slower than the Z-B chimera band in lane 5, and the whole band in lane 6 was stagnated in the loading tank which was circled in lane 6, and the mobility of the duplex band with protein ZBP-1 in lane 10 was the same as the duplex band in lane 9, indicating that the Z-B chimera has combined with the ZBP-1 and the B DNA duplex has not combined with the protein, so we can get the conclusion that we had obtained Z-B chimera. However, part of the band of circle β with it’s complementary linear α added 12μΜ protein ZBP-1 in lane 8 was also stagnated in the loading tank which was circled in lane 8 and part of the band moved synchronously with the circle B DNA band in lane 7, so we guessed that because the sequence we designed consists of APP sequence and when ZBP-1 combined with the sequence, part of the circle B DNA may be changed the structure into Z DNA, which leaded to the result as if ZBP-1 also combined with B DNA.
We used RT-PCR to measure the Tm and we got the data that the Tm of 63nt Z-B chimera is 63.6℃ under the condition of 10mM MgCl2, lower than double strand B-DNA circle (76.6℃) and B-DNA duplex (79.9℃). The data indicated that Z-B chimera can form below the temperature of 50℃.
2. The transcription of Z-B DNA chimera
Figure 4 a is the 8% Native PAGE and figure 4 b is the 14% denaturing PAGE pattern. All the condition of the experiment and operations were the same as experiment of 74nt except the condition of denaturing gel. We used 14% denaturing PAGE with methanamide to make the product denative completely. The formation of Z-B DNA chimera was obtained from the image. So the next step is to transcript.
Figure 5 a is native PAGE pattern and figure 5 b is denaturing PAGE pattern. In figure 5 a, the samples were loaded in the same volume on the 8% native gels for characterization of the transcription product of DNA duplex, lane 4 and lane 5 were to verify whether the duplex can transcript, and we can get the conclusion that the sequences we designed had no problems because the duplex can transcript, there were remaining bands of transcription product of duplex in lane 5 after adding DNaseⅠto the sample in lane 4. In figure 5 b, the samples were loaded in the same volume on the 8% denaturing gels for characterization of the final product we expect, in lane 4, lane 6 and lane 8, we added RNase A to the sample of lane 3, lane 5 and lane 7 respectively and so the disappeared bands were the transcription product and the remaining bands were the material DNAs. We can see that the transcription product in lane 7 moved much slower than the product in lane 3 and lane 5, the mobility was significantly different and the result indicated that the transcription product of Z-B DNA chimera has been obtained.
3. Formation of Z-B RNA chimera:
We didn’t finish this part of experiments, but we are still exploring the method to form Z-B RNA chimera and we believed that we will make it on the base of formation of Z-B DNA chimera.
1. Changing the experiment condition when using native gels to prove the formation of Z-B DNA chimera.
At the beginning of the 42nt experiment, we just use traditional 8% native gel. However, we found that the efficiency of forming Z-B DNA chimera was very low (figure 6), in figure 6 a lane 5, except the band of Z-B DNA chimera, there were still circle α and circle β band left. According to the research that Z-DNA is easy to form under the condition of high concentration of positive ion and considering that heat will be released when the gels running and the structure of Z-B DNA chimera may be destroyed, we added MgCl2 to the native gel and 1×TBE buffer, then added ice to electrophoresis tank to reduce the temperature. Finally, the efficiency of forming Z-B DNA chimera improved a lot and there was only one band of Z-B DNA chimera in figure 6b lane 5.
2. Exploration of the effect of using different concentration of NEBuffer when forming Z-B DNA chimera.
What’s more, we also explored that whether the yield will change if we changed the concentration of the buffer when we prepare Z-B chimera. So we prepared another four kinds of concentration (0.6×, 0.4×, 0.2× and 0.1×) except 1×, and used native PAGE to prove the result (figure 7). The result indicated that there was little differences in yield between different concentration of the bands. The length of DNA we used was 63nt long.