DESIGN

Design

In this experiment, DNA and RNA are our experimental material. In order to make them form left handed DNA (Z-DNA), we designed the DNA sequence by our idea.Preparation of circular DNA is the key point, so all these sequences will be treated particularly (figure.1). From the previous lectures, we can know that the alternating pyrimidine-purine sequence can form left-hand DNA [1]. The classic Z-DNA sequence is d(CG). So we designed three kinds of sequences around Z-DNA to perform different experiments.



Part 1: Preparation of circular DNA



In this part, our goal is to form Z-DNA. The DNA we designed has two complementary mini circles and contains the Z-DNA formation regions with APP (alternative purine-pymidine) sequence (d(TGCG)n) (Fig. 2). The APP sequence is believed to be capable of forming Z-DNA.

In addition to the two complementary ssDNA, we also need to design two DNA splints, which is used for preparation of ssDNA circles (Fig. 2). In the design process of DNA sequence, the software we used is Primer Premier, which is used to detect the mismatch of the DNA sequence. Then we adjusted the sequence according to the results of the software to minimize the number of mismatch, which can improve the yield of the experiment. The ratio of Z-DNA sequence to B-forming sequence is about 12: 10.5. Because the B-DNA double helix has 10.5bp per complete turn and Z-DNA double helix has 12bp per complete turn. We have designed five kinds of DNA sequence (42bp, 53bp, 63bp, 74bp, 84bp) (Table.1). These sequences have two necessary things in common. Firstly, the percent of GC (GC%) is less than 50%; Because high GC% will make it difficult to unscrew the double helix. Secondly, the Tm of splint should be more than 25℃ to make it combine to the DNA linear more firmly.



DNA linear Sequence splint
42 bp linear α 5'AACCGTGCGTGCGTGCGGATCAACTAATACGACTCATCATAA-3' 5'-CACGGTTTTATGAT-3'
linear β 5'-TTATGATGAGTCGTATTAGTTGATCCGCACGCACGCACGGTT-3' 5'-ATCATAAAACCGTG-3'
53 bp linear α 5'-ACGGTACGGGATATAATACGACTCACTATATGCGTGCGTGCGATTCCACCAGA-3' 5'-TACCGTTCTGGT-3'
linear β 5'-TCTGGTGGAATCGCACGCACGCATATAGTGAGTCGTATTATATCCCGTACCGT-3' 5'-ACCAGAACGGTA-3'
63 bp linear α 5'-ATGTAATCTAACTGTGCGTGCGTGGACTAATA CGACTCACTATAAACGGACCAACTATTGAAA-3' 5'-GATTACATTTTCAATA-3'
linear β 5'-TTTCAATAGTTGGTCCGTTTATAGTGAGTCGTATTAGTCCACGC ACGCACAGTTAGATTACAT -3' 5'-TATTGAAAATGTAATC-3'
74 bp linear α 5'-CACCTCGCTTTCAACTTACAA TGCGTGCGTGCGGTCTAATACGACT CACTATAAATCGAACATCAGCAAACGGA-3' 5'-GAGGTGTCCGTT-3'
linear β 5'-TCCGTTTGCTGATGTTCGATTTATAGTGAGTCGTATTA GACCGCACGCACGCATTGTAAGTTGAAAGCGAGGTG-3' 5'-AACGGACACCTC-3'
84 bp linear α 5'-CCTACCACCTCGCTTTCAACTTACAATGCGTGCGTGCGGTCTAA TACGACTCACTATAAATCGAACATCAGCAAACGGACAGAG-3' 5'-GGTAGGCTCTGT-3'
linear β 5'-CTCTGTCCGTTTGCTGATGTTCGATTTATA GTGAGTCGTATTAGACCGCACGCACGCA TTGTAAGTTGAAAGCGAGGTGGTAGG-3' 5'-ACAGAGCCTACC-3'
Table 1. The sequence of Z-DNA. The underlined part of sequence is the APP sequence.

Part 2: The design of the transcription sequence



Our experimental results show that most of our sequences can successfully form Z-DNA DNA. As it is known to us all, these is a close connection between the Z-DNA and transcription [2]. Then we redesigned a 92bp DNA sequence for transcription (Table.2). So we added the T7 promoter sequence include the promoter upstream and downstream sequence to this sequence (Fig. 3). Because the shorter sequence is harder to form Z-DNA according to the previous experimental results, we designed the sequence that about half of the formed dsDNA has Z-DNA conformation and left part has B-DNA conformation, instead of all the alternating pyrimidine-purine sequence. Because too long Z-DNA sequence will make it very stable and isn't helpful for transcription. The software we used in this design process is The mfold Web Server, which can view the secondary structure of the sequence (Fig. 4A). We can change the order of base according to the secondary structure to reduce the stability of the secondary structure.





DNA linear Sequence splint
92 bp linear α 5'AGCAGATATAATACGACTCACTATAGGGAAGC AGGATTGAACAAGATTGATGTGTGTACGTGA TGGAGTAGGTCAAA5'- TCTGCTGTACAC-3'-3' 5'-CACGGTTTTATGAT-3'
linear β 5'-TTATGATGAGTCGTATTAGTTTGACCTACTCCATCACG TACACACATCAATCTTGTTCAATCCTGCTTCCCTATAGTGAG TCGTATTATATCTGCT-3' 5'-ATCATAAAACCGTG-3'
Table.2 The sequence of Z-DNA. The red part is the T7 promoter sequence. The yellow part is the raised A/T. The blue part is the APP sequence.


Part 3: The design of the Z-RNA sequence

We not only thought of the Z-DNA, we also took the Z-RNA into consideration, we thought about whether the RNA can form the Z-DNA structure by the same way. What's more, we are interested in the function of the small interfering RNA (siRNA), which plays an important role in the process in which genes can be silenced sequence-specifically [3]. So we had changed the experimental material from DNA to RNA (Table.3) and add the siRNA sequences into the dsRNA circle. The two siRNA sequences we selected are the specific E6 and E7 siRNA for treatment of HPV16 positive cervical cancer [4], one of which contains the APP sequence and is added in the Z-DNA part. The other without the APP sequence is added in the B-forming part (Fig.5). Because the APP sequence is very easy to form stable secondary structure according to the results from The mfold Web Server (Fig. 4B), which is harmful to make the RNA become a circle. We design a special kind of DNA sequence called accessorial DNA, which can combine with the alternating pyrimidine-purine sequence and avoid generating stable secondary structure (Fig. 4C).



DNA linear Sequence splint(12bp) splint(16bp)
65 bp linear α 5'-UCUGGACCGGUCGAUGUAUGUC UUGCACACGUGUACUCUUAAGC AACAGUUACUGCGACGUGAAA-3' 5'- TCTGCTGTACAC-3' 5'-GGTCCAGATTTCACGT-3'
linear β 5'- UUUCACGUCGCAGUAACUGUUGC UUAAGACUACACGUGUGCA AGACAUACAUCGACCGGUCCACA -3' 5'- GTGTACAGCAGA-3' 5'-ACGTGAAATGTGGACC-3'
A accessorial DNA-1 5'-CACGTGTGCAAGACATACATCGAC -3'(24bp)
B accessorial DNA-1 5'-GTCTTAAGCAACAGTTAC -3' (18bp)
B accessorial DNA-2 5'-CGATGTATGTCTTGCACAC-3'(19bp)
Table.3 The sequence of Z-DNA. The blue part is the B-forming sequence. The red part is siRNA sequence. The green part is the Z-DNA sequence.


References

  1. Gary P. Schroth, Ping-Jung Chou, and P. Shing Ho. (1992) Mapping Z-DNA in the Human Genome [J]. Journal of Biological Chemistry 267, 11846-11855.
  2. Liu L F, Wang J C. Supercoiling of the DNA template during transcription [J]. Proc Natl Acad Sci U S A, 1987, 84(20):7024-7027.
  3. Raymond M. Schiffelers, Jun Xu, Gert Storm, Martin C. Woodle, andPuthupparampil V. Scaria. (2005) Effects of Treatment With Small Interfering RNA on Joint Inflammation in Mice With Collagen-Induced Arthritis [J]. ARTHRITIS & RHEUMATISM 52, 1314-1318.
  4. K Yamato, T Yamada, M Kizaki, K Ui-Tei, Y Natori, M Fujino, T Nishihara, Y Ikeda, Y Nasu, K Saigo and M Yoshinouchi. (2008) New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer [J]. Nature 15, 140-153.