Revision as of 18:59, 21 October 2019

Strand 1 (free aptamer) for in vivo synthesis of DNA nanostructure

The biobrick design allows the use of golden gate assembly to insert any aptamer sequence onto the 5' end of strand 1 ssDNA.

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

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 .

This part is inserted with a special site flanking by 2 Bsal RE sites which could be edited into different aptamer sequences and hence increase the flexibility of the design of our nanostructure.

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]

Characterization

NDC-MA

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

1. ↑ 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.
2. ↑ 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
3. ↑ 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.
4. ↑ Kumar, V., Palazzolo, S., Bayda, S., Corona, G., Toffoli, G. & Rizzolio F. (2016). DNA Nanotechnology for Cancer Therapy. Theranostics, 6(5), 710-725.
5. ↑ Sun, G. & Gu, Z. (2015, January 26). Engineering DNA Scaffolds for Delivery of Anticancer Therapeutics. Biomaterials Science, 3(7), 1018-1024.