Transformation of information
Although the stem loop can work as a “lock” to protect our ciphertext from being stolen, information burglar also can decode it by sequencing DNA directly. To overcome this situation, we were inspired by RNA splicing, a critical mechanism by which to modify transcriptome, it also can be used to realize the “misleading” part of the project. In our case, specific sequences contain misleading words are inserted into the information sequence we just designed by the code book as intron. Therefore, only after receivers tranform the “key plasmid” can cryptographic information be transcripted and spliced normally, and can the receiver get the correct information. As for information burglar, he will make a normal DNA sequncing only to obtain a misleading information with wrong meaning. Therefore, pre-mRNA splicing become a new method protecting our ciphertext from being decoded.
Information expression verification
Yeast: a-type yeast.
Function: Using the specific “key-lock” interaction, information can be obtained when expressing a specific key.
Vector construction: Gene fragments of information with a stem loop and Ura screening genes were inserted between the two homologous arms, HA1 and HA2 to construct the pCYC-stem loop-information-Ura plasmid. HA1 and HA2 act as homology arms to promote the homologous recombination, which allows the integration of the DNA containing the stem loop lock sequence into the yeast genome. And the constructed plasmid is shown in Figure 1.
Figure 1： pCYC-stem loop-Information-Ura
Verification of homologous recombination
Information fragments with homology arms were transformed into yeast. In order to ensure that the information fragments were successfully integrated into the yeast genome, we picked the monoclonal culture and extracted the DNA for PCR verification. The results demonstrate that homologous recombination is successful（Figure 2）.
Figure 2： Results of PCR validation of homologous recombination.
Access to information
Then we extracted the total RNA of yeast and carry out reverse transcription. DNA sequencing will be used to verify the correct expression of the information fragment is expressed, which will be compared with the original information. The sequencing results back from the sequencing company showed that our information fragments were correctly expressed. And in the end we got the right information (Figure 3).
Figure 3: The sequencing results and information obtained nucleic acid to convert word.
At first we decided to use false information as an intron. In this way, though the thief gets the information sequence from the yeasts, he will get sentences with completely different meanings. And only the mature RNA that has been spliced will carry the correct information. Therefore, the only way that we can get the correct meaning of the information is to reserve transcript the mature RNAs after getting it. However, after checking the sequences, But we don't have much time to try out whether the introns we design can be spliced, so we choose to use the naturally occurring introns in yeast. Unfortunately, our final result showed that the splice did not happen, either. So this is also the direction of our team's future efforts.
However, after checking the sequences, But we don't have much time to try out whether the introns we design can be spliced, so we choose to use the naturally occurring introns in yeast. Unfortunately, our final result showed that the splice did not happen, either. So this is also the direction of our team's future efforts.