Difference between revisions of "Part:BBa K2054001:Design"

Revision as of 02:42, 30 October 2016

Oligo 1

Design Notes

This device was modified from a recent publication about in vivo synthesis and self-assembly of DNA nanostructures. We made use of the technique described in the article to synthesize oligos for our tetrahedral nanostructure, where the sequences are generated by a software called Tiamat. This device will enable the single-stranded DNA oligo 1 to be synthesized inside cells. The ssDNA oligos produced can be purified and used to form our tetrahedral nanostructure, which consists of 5 oligos.

Below show our tetrahedral nanostructure and the sequence of the oligos.

One of the key feature of our tetrahedral nanostructure is the strand displacement induced by the input strand. The schematic diagram below illustrates how.

NB: for simplicity, only the active component (i.e. O1+O5 beacon is drawn)

Below is a 12% polyacrlamide gel to show the assembly (lanes 2 to 7) and the strand displacement (lanes 6, 8-10) of the tetrahedron. Lanes 2, 3, 4, 5 and 6 correspond to the individual single-stranded oligos 1, 2, 3, 4 and 5, and lane 6 has all the five oligos put together in the thermocycler, where the tetrahedron is expected. Lanes 6, 8 and 9 contains oligo 5, input strand and the dimer of O5 and input, the expected displaced product. Finally, lane 10 shows the addition of input strand to the tetrahedron, inducing the strand displacement.

Construction and production of ssDNA The design is based on the literature mentioned and contains:

a strong promoter BBa_J23100 from the Registry of standard biobricks a ‘r_oligo’ region that contains the sequence of our desired oligos and more (see below); a terminator BBa_B0054, which is also from the Registry;

The ‘r_oligo’ region will transcribe a product that contains a non-coding RNA (ncRNA) and a HIV-Terminator-Binding Site (HTBS) that exhibit a 3’-hairpin structure:

The HTBS serves as a terminator in this gene, where the HIV reverse transcriptase binds. During the reverse transcription, the binding of HIVRT initiates the elongation, which is aided by another RT murine leukemia reverse transcriptase (MLRT). RNase H then cleaves specifically the ncRNA-DNA linkages, which leaves the desired ssDNA to hang, but still attached to the HTBS on its 5’ end. RNase A then breaks to release the desired ssDNA. The following diagram summarizes the in vivo conversion.

Source

Plasmid Construction: Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.

Oligo Construction: Nakatsuka, K., Shigeto, H., Kuroda, A., & Funabashi, H. (2015). A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids. Biosensors and Bioelectronics, 74, 222-226.

References

Plasmid Construction: Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.

Oligo Construction: Nakatsuka, K., Shigeto, H., Kuroda, A., & Funabashi, H. (2015). A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids. Biosensors and Bioelectronics, 74, 222-226.