Part:BBa_K3063041
Strand 4 (EpCam) for in vivo synthesis of DNA nanostructure
The biobrick design allows the in vivo production of Strand 4 ssDNA of DNA nano drug carrier with EpCam (cancer stem cell surface marker aptamer) attached at the 5' end.
The biobrick contains a promoter, a single strand DNA(ssDNA) production region, reverse transcriptase binding site (HTBS) and also a terminator. The promoter, ssDNA, HTBS site were constructed in a seamless way to allow correct ssDNA sequence after reverse transcription. The biobrick is used together with HIV reverse transcriptase (HIVRT) and Murine Leukemia Reverse Transcriptase (MLRT) co-expressed in E. Coli cells. It allows in vivo-synthesis of ssDNA due to the reverse transcription, and also RNA template degradation function of reverse transcriptases. The ssDNA produced could anneal to produce DNA nanostructure in vivo.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 13
Illegal NheI site found at 36 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
Biology
Part structure
This biobrick is part of a dual tumour-specific drug delivery system with Salmonella Typhi we designed. Any strand 1 could combine with any strand 2, 3 and 4 and encodes four ssDNA each with an aptamer of different function attached at the 5’ end. These 4 ssDNA can be assembled into a DNA tetrahedron with each vertex consists of one desired aptamer .
In each biobrick, the component strand encoding region is followed by an HTBS, a stemloop for binding of HIV reverse transcriptase and B0054, a strong terminator. For our design to function, these three structural components must be together as a basic part. This part design is modified from a previously described method of ssDNA synthesis using this HTBS sequence and B0054 terminator.[1] Each component strand encoding region is originally designed, with component strand sequence generated by 3D nanostructure simulation in the software TIAMAT. [2]
Therapeutic DNA nanostructure
Design principles: safety, efficient cell entry, flexibility, stability
In this project, a DNA nanostructure, namely Nano Drug Carrier with multiple aptamers (NDC-MA) for linking Salmonella Typhimurium and targeting liver cancer cells were designed and tested. This nanostructure is made up of 4 single-stranded DNA synthesized using 4 different BioBricks.
Our Nano Drug Carriers are composed entirely of DNA, which is non-toxic and degradable inside human cells. The Nano Drug Carriers are designed as tetrahedrons to facilitate cell entry. Previous studies have shown that three-dimensional DNA nanostructure enter mammalian cells more efficiently than two-dimensional AS1411 or linear structures. [3]Tetrahedron is chosen because we consider it the simplest three-dimensional structure, which can be easily assembled from just a few DNA strands. Building the Nano Drug Carrier with separate DNA strands means that much flexibility is allowed for functional modifications. Functional DNA sequences can be conveniently added to the 4 vertices of the tetrahedron to achieve oligonucleotide delivery or cell antigen binding, enhancing the effect of the drug. Three-dimensional structures composed of double stranded DNA have been shown to be stable in extracellular compartment, making them ideal as drug carriers.[4]
Our NDC-MA for liver cancer therapy can be conveniently assembled by annealing the 4 single-stranded DNA: 4 strands each form one face of the tetrahedron with one aptamer on each vertex. Cancer therapy drug, doxorubicin (Dox) can be loaded onto the tetrahedron by DNA intercalation.[5]
The NDC-MA we designed are expected to have multiple functions: 1) Specifically recognising and targeting cancer cell; 2) Specifically recognising and targeting cancer stem cells and 3) Attaching to the surface of Salmonella Typhimurium with the help of 2-4 different aptamers: AS1411, SYL3C, ST1 and APT33.
Aptamer 1: ST1
With the help of a technique called SELEX[6], a number of aptamers targeting antigen of interest was selected and could be use for therapeutics. ST1, is an aptamer discovered in this way.[7] ST1 recognise and bind to the surface antigens of Salmonella Typhimurium. With this aptamer at the vertex, our therapeutic nanostructure could link with the surface of Salmonella Typhimurium and hence facilitate the transfer to tumour region.
Aptamer 2: APT33
APT33 is also an aptamer targeting Salmonella surface antigens. The chance of nanostructure linking Salmonella is highly increased with both ST1 and APT33. [8]
Aptamer 3: AS1411
AS1411 is an aptamer recognising and binding nucleolin on cell surface.[9] Since there is an over-expression of nucleolin on cancer cells, this aptamer could preferentially target and bind to cancer cell surface, making it a perfect aptamer to attach to our therapeutic nanostructure for specific binding.
Aptamer 4: SYL3C
SYL3C is an aptamer recognising and targeting the marker on cancer stem cell, EpCam (Epithelial cell adhesion molecule).[10]Cancer stem cell is considered to be the core of tumorigenesis and cancer cell differentiation. With our nanostructure intercalating with Doxorubicin, we wish to reduce the stemness of the cancer stem cell and inhibit tumour growth.[11]
Characterization
NDC-MA
To test the feasibility of our designs, chemically synthesized DNA oligos of the sequences generated by Tiamat were used to assemble the NDC-MA. Native polyacrylamide gel electrophoresis (PAGE) was used to visualize the component strands and the assembled NDC-MA.
Proof by PCR
The strands was synthesised by the ETHERNO system, an E. coli-driven in vivo ssDNA synthesis system we developed last year. The first eight lanes contain duplicates of each strand amplified by a strand-specific forward primer and a backbone-specific reverse primer. The remaining lanes are duplicates of each strand amplified by a backbone-specific forward primer and a strand-specific reverse primer.
Proof by Anealing
The strands was also synthesised by the ETHERNO system and another method was used to verify them. Strand 4 is tagged by a fluorophore and annealed with the other strands. Since all strands combine together and form a tetrahedron, each strands could complementarily bind to each other. Lane 2, 3, 5 and 7 are ssDNA and lane 4, 6 and 8 are different strands annealed to strand 4. The size confirms the successful synthesis of the ssDNA.
Parts application
Transmission Electron Microscopy
Our DNA tetrahedral structures are stained and visualised by a Philips CM 100 Transmission Electron Microscope with 100 kV operating voltage.
Since the 3' end of each strand is modified to have a thiol group, gold nanoparticles could then be attached to it. After the strands assemble, all four vertices of the final tetrahedron will be attached by a gold nanoparticle and could be observed under the Transmission Electron Microscope.
References
- ↑ Elbaz, J., Yin, P. & Voigt, C.A. (2016, April 19). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nat Commun. 7:11179.
- ↑ Williams, S., Lund, K., Lin, C., Wonka, P., Lindsay, S., & Yan, H. (2009). Tiamat: A three-dimensional editing tool for complex DNA structures. In Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) (Vol. 5347 LNCS, pp. 90-101). (Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics); Vol. 5347 LNCS). DOI: 10.1007/978-3-642-03076-5_8
- ↑ Xia, Z., Wang, P., Liu, X., Liu, T., Yan, Y., Yan, J....He, D. (2016, March 8). Tumor-penetrating peptide-modified DNA tetrahedron for targeting drug delivery. Biochemistry, 55(9),1326-1331.
- ↑ Kumar, V., Palazzolo, S., Bayda, S., Corona, G., Toffoli, G. & Rizzolio F. (2016). DNA Nanotechnology for Cancer Therapy. Theranostics, 6(5), 710-725.
- ↑ Sun, G. & Gu, Z. (2015, January 26). Engineering DNA Scaffolds for Delivery of Anticancer Therapeutics. Biomaterials Science, 3(7), 1018-1024.
- ↑ Tuerk, Craig, and Larry Gold. "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." science 249.4968 (1990): 505-510.
- ↑ Park, Hae-Chul, et al. "Development of ssDNA aptamers for the sensitive detection of Salmonella typhimurium and Salmonella enteritidis." Applied biochemistry and biotechnology 174.2 (2014): 793-802.
- ↑ Wang, Bin, et al. "Label-free biosensing of Salmonella enterica serovars at single-cell level." Journal of nanobiotechnology 15.1 (2017): 40.
- ↑ Kang, Won Jun, et al. "Multiplex imaging of single tumour cells using quantum‐dot‐conjugated aptamers." Small 5.22 (2009): 2519-2522.
- ↑ Liu, Jin-Xia, et al. "Nonenzymatic amperometric aptamer cytosensor for ultrasensitive detection of circulating tumor cells and dynamic evaluation of cell surface N-Glycan expression." ACS Omega 3.8 (2018): 8595-8604.
- ↑ Saygin, C., Matei, D., Majeti, R., Reizes, O., & Lathia, J. D. (2018). Targeting cancer stemness in the clinic: from hype to hope. Cell Stem Cell.
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