In the experiment process, we need to design a double ring with a cross structure, and can realize the opening of the cross structure. This requires us to understand the energy relationship between unopened and opened stem loop sequence, so as to better complete the design of stem loop. Based on this, we need to design a class of cross structure sequence and its corresponding no-stem ring sequence, and predict its TM value by software, and thus explore the relationship between them.
In order to better simulate the double-stranded stem loop sequence, we designed a circular DNA sequence, using 10bp (CG) N sequence at both ends of the stem loop sequence, and using 10bPC-G base pair pairing to simulate the infinite double-stranded structure at both ends in the natural state.
At the same time, when designing stem loop sequence, we found:
- - In vivo, there is no cross structure sequence that forms stem rings simultaneously and can complement each other.
- - In vivo, it is easier to generate cross sequence if the ring structure with -GNNA - and -CNNT- as stem ring.
Based on this, we designed a single loop sequence: a continuous 10bp (CG) N sequence was used at both ends of the cross structure to ensure the stability of the cross sequence; At the same time, in order to simulate the energy model of the cross structure sequence as realistically as possible, we designed the two stem rings into the same structure to simulate the formation of the cross structure. In the control group, the two stem rings were designed as complementary sequences to simulate the opening of the cross sequence.
Figure 1. the cross structure sequence
Figure 2. the control group simulates the opening of the cross sequence
Finally, after determining the basic structure of the cross sequence, we use software to determine the stem-loop structure required for modeling and the corresponding control structure based on the principle of reducing the normalized whole.
Results and Discussion
Based on the principle of reducing the normalized whole, we designed stem-ring structures with length of 8,9,10,11 and 12bp and their corresponding control structures respectively. The corresponding energy values are predicted by MFOLD software.
Figure 3. The corresponding energy values of stem-loop sequence
We found that the overall TM value of cross sequences was slightly higher than that of the control group. The TM values of both decreased with the increase of sequence length, which was consistent with our expected results.