Other

Part:BBa_K2592000

Designed by: Janice Law   Group: iGEM18_Hong_Kong_HKU   (2018-09-23)


Strand 1 for in-vivo synthesis of Nano Drug Carrier

This part encodes a component strand of our originally designed Nano Drug Carrier (NDC). The strand-encoding region is followed by an HIV-terminator binding site (HTBS). This component strand can be expressed in the presence of HIV reverse transcriptase and murine leukaemia virus reverse transciptase inside E. coli DH5alpha (Elbaz, 2016).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 13
    Illegal NheI site found at 36
    Illegal NheI site found at 46
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Introduction

Biology

Part structure

This biobrick is part of a DNA nanostructure production system we named ETHERNO (E. coli-synthesized Therapeutic Nanostructures). Each of the biobrick submitted encodes a single-stranded DNA of specific sequence. These ssDNA synthesized can then be extracted for assembly of our originally designed therapeutic 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]

For nanostructures composed of multiple DNA strands of different sequences, the BioBricks required follow the same basic structure as shown in Fig. 1.

Figure 1. ETHERNO workflow

Therapeutic DNA nanostructure

Design principles: safety, efficient cell entry, flexibility, stability

In this project, 2 DNA nanostructures, namely Nano Drug Carrier (NDC) and Nano Drug Carrier-AS1411 (NDC-AS) for breast cancer therapy were designed and tested. Each nanostructure is made up of 5 single-stranded DNA synthesized using 5 different BioBricks. The 2 nanostructures have 2 componant strands in common, so a total of 8 BioBricks were made and submitted.

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]

Figure 2. NDC
Our NDC (Fig. 2) for breast cancer therapy can be conveniently assembled by annealing 5 single-stranded DNA: 4 core strands forming a tetrahedron and 1 strand (Strand 5) to be displaced by target miRNAs, miR21 and miR217 inside breast cancer cells.[5] Part of Strand 5 is a displacement toehold complementary to miR21. The 5' segment next to the toehold is complementary to another target, miR217. The 3' segment binds complementarily to 5' end of Strand 1. Breast cancer drug, doxorubicin (Dox) can be loaded onto the tetrahedron by DNA intercalation.[6] Therapeutic functions are expected to be exerted in two ways: cancer miRNA down-regulation by the displaced strand and intracellular release of doxorubicin.


Figure 3. NDC-AS
Inspired by the Human Practices interview with molecular pathologist Dr. Lau, NDC-AS (Fig. 3) was designed for more cell-specific drug delivery. AS1411, a previously characterized DNA aptamer against nucleolin was incorporated into Strand 2 of NDC. [7]As nucleolin is overexpressed in breast cancer cells, NDC-as was expected to preferentially enter cancer cells and demonstrate better entry and cancer cell cytotoxicity than NDC.[8]The complimentary region between the tetrahedral base and the strand to be displaced was also extended to increase displacement specificity. Extension was done by adding TCG/AGC repeats to facilitate doxorubicin intercalation.


Characterization

NDC

To test the feasibility of our designs, chemically synthesized DNA oligos of the sequences generated by Tiamat were used to assemble the NDCs. DNA-21 and DNA-217, which are DNA equivalents of miR21 and miR217 were initially used as the inputs, before using RNA. Native polyacrylamide gel electrophoresis (PAGE) was used to visualize the component strands and the assembled NDCs.



Figure 4.PAGE (8%) image of NDC component single strands, DNA inputs and assembled NDC

After assembly, component strands were seen to have formed complexed too large to be run through the gel PAGE (Fig.4). These large complexes close to the loading well were expected to be successfully assembled NDC that could not pass through the gel due to its large 3D structure, with a tetrahedral base of around 16nm per edge. The band of around 20bp seen below NDC were unbound Strand 5, which was designed to be only partially complementary to Strand 1.



Figure 5. PAGE (8%) images showing annealed strands of various combinations. Three-dimensional NDCs are seen to be of much a larger band size than incomplete two-dimensional structures.

To confirm the assembly of NDC from the 5 component strands, PAGE of structures formed by different strand combinations was done (Fig.5). After annealing, component strands formed complexes larger than their individual sizes, proving DNA structure assembly due to base complementarity. Large complexes close to the bottom of wells could only be seen in samples containing Strand 1, 2, 3 and 4, which together form the 3D tetrahedral base of NDC. These results supported the successful formation of a specific 3D DNA structure.



Figure 6. PAGE (8%) showing strand displacement by DNA-21 and DNA-217 after 30 minutes incubation at 37°C. Displacement of Strand 5 out of NDC by the targets are visualized as bands of size 40 to 50bp in lane 3 to lane 5. Table shows relative band intensities compared against expected Output for preliminary displacement efficiency evaluation.

Our NDC was designed to release to an anti-microRNA oligonucleotide upon binding with intracellular miR21 and miR217. As a preliminary test of strand displacement efficiency, we incubated DNA-21 and DNA-217 with NDC in phosphate buffered saline at 37C and ran a page (Fig.6). Successful strand displacement is shown, because when displaced by 2 inputs, a band of the correct output size was produced and the band intensity, correlated with DNA concentration, was higher than when displaced by either 1 of the inputs.



Figure 7. PAGE (8%) showing strand displacement by DNA-21, 21-5' mut and 21-3' mut after 30 minutes incubation at 37°C. Lane 5 is the expected output, which is also present in Lane 3 showing correct displacement. Lane 6 and 7 shows sub-optimal binding of the mutants to Strand 5, similar to the outputs in Lane 4 and 5.

The specificity of strand displacement was tested using two mutant DNA inputs, 21-5’ mut and 21-3’ mut, each carrying a 6-base mutation at its 5’ or 3’ end respectively. As visualized on the PAGE image, both 21-5’and 21-3’ mut failed to fully displace Stand 5 to produce the expected output, leaving unbound Strand 5 and inputs as bands of around 30bp and 20bp respectively. The smeared output produced by 21-5’ mut could be explained by its interaction with Strand 5 at sequences outside the expected binding region.




Parts application

The collected single-stranded DNA (ssDNA) were suspended in water which contained leftovers of chemicals used in extraction. Because of this, these ssDNA could not be easily visualized on PAGE, as performed above for NDC assembled from chemically synthesized ssDNA. None the less, we tried to amplify the ssDNA extracted using primers that overlapped with the component ssDNA sequences. We expected to get back bands of the expected ssDNA sizes as shown in Fig.1 and Fig.6 on PAGE. We also annealed the strands synthesized by ETHERNO, to see if large complexes similar to NDC and NDC-AS as shown in Fig.1 and Fig.6 could be produced.

Products of ETHERNO

Expected bands of Strand-1, Strand 1-AS, Strand 5 and Strand 5-AS can be seen in Fig.20a, which means these 4 strands were present in the ssDNA extracted from ETHERNO. Other strands could not be amplified here, may be due to problematic primer design. Designing primers for these nanostructure component strands was challenging due to the complicated secondary structures formed. Since these extracted ssDNA were able to form large structures that could not pass through PAGE.(Fig. 20b), we moved on to look for any successfully assembled NDC and NDC-AS under transmission electron micrpscopy.

TEM of NDC made by ETHERNO
TEM of NDC-AS made by ETHERNO

Cloning

Colony PCR to check presence of parts

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. ↑ Singh , R. & Mo, Y. (2013, March 1). Role of microRNAs in breast cancer. Cancer Biol Ther, 14(3), 201–212.
  6. ↑ Sun, G. & Gu, Z. (2015, January 26). Engineering DNA Scaffolds for Delivery of Anticancer Therapeutics. Biomaterials Science, 3(7), 1018-1024.
  7. ↑ Bates, P.J., Laber, D.A., Miller, D.M., Thomas, S.D. & Trent, J.O. (2009, June). Discovery and Development of the G-rich Oligonucleotide AS1411 as a Novel Treatment for Cancer. Experimental and Molecular Pathology, 86(3), 151–164.
  8. ↑ Fonseca, N.A., Rodrigues, A.S., Rodrigues-Santos, P., Alves, V., Gregório, A.C., Valério-Fernandes, A.... Moreira, J.N.(2015, November). Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination. Biomaterials, 69, 76-88.
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