Part:BBa_K2202000:Design

ssDNA for producing in-vivo tetrahedral structure Strand1

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 182
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 101
Illegal SapI site found at 272

Design Notes

This sequence is a modification of the parts BBa K2054001,BBa K2054002, BBa K2054003 ,BBa K2054004 , BBa K2054005 from the iGEM Hong University Team 2016 team. This year our team is produced a Pre-tetrahedron structure which folds upon binding to the Huntington's disease biomarker Hsa-miR-34b ^[1]. The Parts BBa K2202000,BBa K2202001,BBa K2202002,BBa K2202003,BBa K2202004 work together to form the nanostructure. The sequence was generated using a DNA nanotechnology software called Tiamat ^[2]

Shown below is the schematic of how the Pre-tetra nanostructure folds only upon binding to the target to produce a G-quadruplex and hence we expect the signal to noise ratio to improve from last year's nanostructure.

Fig 1: Schematic of the functioning of the nanostructure.

Principle of the Biobrick^[3,4]

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.

Gel Images

The gel bands of the individual oligonucleotides allow for the estimation and verification of the sizes of the oligonucleotides by comparison with the DNA ladder. The formation of the 3-dimensional structure from the 2-dimensional DNA nanostructure in the presence of the specific target can also be clearly observed from the above gel image as a prominent shift from the gel band in lane 9 to the gel band in lane 10 can be distinctly seen.

Fig 2: PAGE gel (8%, 70V) showing bands of individual oligonucleotides (O1-O6) of DNA nanostructure, along with the target, 2-dimensional nanostructure without the presence of target (pre-tetra) and 3-dimensional nanostructure after detection of the target (tetra).

Source

This Part was generated using a DNA nanotechnology software called Tiamat. This is a computationally designed sequence.

References

1. Gaughwin, P.M., Ciesla, M., Lahiri, N., Tabrizi, S.J., Brundin, P. and Björkqvist, M., 2011. Hsa-miR-34b is a plasma-stable microRNA that is elevated in pre-manifest Huntington's disease. Human molecular genetics, 20(11), pp.2225-2237.

2. Williams, S., Lund, K., Lin, C., Wonka, P., Lindsay, S. and Yan, H., 2008, June. Tiamat: a three-dimensional editing tool for complex DNA structures. In International Workshop on DNA-Based Computers (pp. 90-101). Springer, Berlin, Heidelberg.

3. Elbaz, J., Yin, P. and Voigt, C.A., 2016. Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.

4. Team Registry HKU iGEM 2016:https://parts.igem.org/Part:BBa_K2054001:Design