Part:BBa_K4665170
Octahedron DNA-origami (3024bp long; 42bp edges)
Biology and Usage
DNA origami is a technique that allows for the fabrication of complex nanostructures through guided DNA folding (Majikes & Liddle, 2021). The process involves designing a long ssDNA molecule, referred to as a scaffold, which acts as a backbone for the assembly of defined DNA nanostructures. The ssDNA scaffold will be folded into the desired configuration using short ssDNA fragments called staples. The staples are complementary to specific regions in the ssDNA scaffold and will fold the scaffold strand into the desired structure via complementary base pairing. Many different potential structures of DNA origami can be achieved by altering the staple sequences.
For the purpose of our project, Sublimestone, an octahedral 3D lattice form was chosen that acted as a nucleation site for the second module of our project, calcite mineralization.
The staple sequences provided below were obtained through an algorithm, ensuring the desired octahedral shape is achieved during the folding experiments with the mixture of the ssDNA scaffold and the staples. The octahedral structure was chosen for its thermodynamic stability, resisting disassembly or rearrangement (Julin et al., 2020). After careful analysis, it was determined that triangular structures have the highest mechanical strength (Tandon, 2021). However, the simplicity of the shape would reduce the surface area needed for nucleation. Hence, the octahedron was chosen as the base of the lattice.
The production of ssDNA sequences involves a double transformation process in E. coli using M13-derived helper plasmids and phagemids. The helper plasmid contains all the coding genes of the M13 phage, excluding the M13 origin of replication and regulatory sequences necessary for phage replication (Behler et al., 2022). In contrast, the phagemid contains the M13 origin of replication, M13 phage regulatory sequences, and a user-defined scaffold sequence (Behler et al., 2022). When both the helper plasmid and phagemid are present simultaneously within the bacterial cell, the helper plasmid provides the cell with the necessary phage machinery for ssDNA production. Simultaneously, the phagemid includes the required regulatory sequences to initiate ssDNA production, with the user-defined sequence serving as a template for ssDNA synthesis.
The phagemid used for this project is the pScaf_3024 phagemid developed by Nafisi et al. (2018). This phagemid has a 3024 bp insert sequence in which the user-defined scaffold template can be implemented. They converted a pUC18 vector into the described phagemid for custom ssDNA production by adding four components: 1) a full-length M13 origin for ssDNA initiation. 2) Kpnl and BamHl restriction sites for insert cloning. 3) M13 packaging signal for phage particle export. 4) modified M13 origin to serve as the ssDNA synthesis terminator.
The helper plasmid used in our project is the HP17_KO7 developed by Praetorius et al. (2017). It contains the coding parts of the M13KO7 helper phage which have been cloned into a pSC101 backbone carrying a kanamycin resistance gene. The M13 origin of replication has been entirely removed, leading to the absence of phage production.
Characterisation
Conversion into ssDNA scaffold
This scaffold sequence must first be converted to a ssDNA sequence before being suitable for folding experiments. In our project, we followed the protocol created by Noteborn et al. (2022). First, the dsDNA scaffold sequence was amplified using a standard primer (5' GGGATTCATGGTGTATTGCTTCACC 3') in combination with a modified primer containing 5 phosphorothioate linkages(5'C*A*T*A*T*GACGCGCCCTGTAGC 3') (ordered from IDT). These linkages are introduced at the first 5 basepairs of the 5' end of the desired scaffold strand. After PCR, the phosphorothioate-modified scaffold can be selectively digested with T7 exonuclease (obtained from New England Biolabs). This enzyme will degrade the strand lacking phosphorothioate linkages from the 5' to the 3' end, while the phosphorothiate-modified strand remains protected against T7 exonuclease digestion.
Figure 1. Gel of ssDNA scaffold (1) Plasmid containing dsDNA scaffold (2) dsDNA scaffold (3) ssDNA scaffold
Well (1) shows the pScaf_3024 plasmid in which the scaffold sequence is incorporated (acquired from Douglas Laboratories through Addgene). In the second well, the dsDNA scaffold resulting from the PCR using the phosphorothioate primer is observed at ~3000 bp. The third well showcases the dsDNA scaffold sequence from well 2 post-T7 exonuclease digestion. Consequently, well 3 should contain the ssDNA scaffold sequence. A very faint band that is slightly shifted downwards is observed, indicating the presence of the ssDNA counterpart of the scaffold sequence.
Folding Experiments
We assessed the folding of the ssDNA scaffold into our target octahedron using diverse conditions as specified in the procedure by Wagenbauer et al. (2017). Three different factors were varied: temperature, MgCl2 concentrations, and staple-to-scaffold ratios.
Figure 2. (1)T1 (2)T2 (3)T4 (4)T5 (5)T7 (6)T8 (7)RM1 (8)RM2 (9)M5 (10)M10 (11)M15 (12)M20 (13)M25 (14)M30
The prominent bands highlighted within the red boxes represent the surplus of staple strands introduced for the folding experiment. The ~3000 bp fragments correspond to the remaining scaffold ssDNA strands that were not included in the folding process. The bands within the wells, indicated by the green box, and some faint bands positioned above the DNA ladder (arrow) suggest the existence of octahedron structures. Octahedron structures are anticipated to have reduced mobility within the gel, hence they are expected to be situated above the 3024 bp band corresponding to the scaffold sequence.
Transmission Electron Microscopy
Negative-Stain Transmission Electron Microscopy (TEM) was performed to visualize the octahedron structures after the folding experiments. The sample preparation was performed by an experienced technician from the M4I institute in Maastricht. Two continuous carbon grids were used per sample for the TEM sample preparation. The continuous carbon grids were glow discharged for 10 seconds and subsequently 10-15µl of the DNA origami sample was applied. This was incubated for 10 minutes and excess liquid was removed with filter paper. The carbon grids were stained with Uranyl acetate and allowed to air-dry for contrast enhancement.
Figure 3.
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Figure 7.
Figure 8.
The TEM images reveal structured DNA formations, hinting at the existence of octahedral DNA structures. However, to reach a conclusive answer, further measurements and subsequent SEM images for three-dimensional visualization would be required.
Staple Sequences
| Row | ID | Type | Sequence |
|---|---|---|---|
| 1 | octa_42nt_A1 | vertex | AGGGCGAAATCTCCAGATTTTTTTGAGTGCTCTT |
| 2 | octa_42nt_A2 | edge | CATTTCTTGATGGCCTCTCAGCATAACCGTCTGGACTCCAACGTCAA |
| 3 | octa_42nt_A3 | vertex | TTAATTGCGGATCCAATTTTTTTTCTTCATTGGC |
| 4 | octa_42nt_A4 | edge | GTTATGGATAAACTTGCGAAAGGTTTCTCCAGTGCAGGGAGTGATCT |
| 5 | octa_42nt_A5 | vertex | ACTTCTTTGCTGCTTGATTTTTTTCCACGTCGAT |
| 6 | octa_42nt_A6 | edge | ACCTGCGCACACATCTTGGTTCAGAGTTCAACAGCTCGTCATACTCC |
| 7 | octa_42nt_A7 | vertex | TCGGACTTCATCTTCGGTTTTTTTTGGTGTGCACTTGGGATAATCTCATA |
| 8 | octa_42nt_A8 | edge | AGGCCCAATGTAGAAACGCAACAGGTGTGG |
| 9 | octa_42nt_A9 | edge | AGAACACCGTTTTTTTTTTTTTTTTTCTCATAGGTGTACTGGAACTCGCTGGACGAGCTTGCTGGCGCAAACATTCTTCC |
| 10 | octa_42nt_A10 | vertex | TGCTGCTGAAGTGCCAGTTTTTTTCTGGACCGTATGGGTTGCGCGCTTCTT |
| 11 | octa_42nt_A11 | edge | TGTGAACTTTGACAGTGTCTTACCGGTGTCACAGACCCCAGTCGTAA |
| 12 | octa_42nt_A12 | vertex | CAGTTCCAACTTGTGCTTTTTTTAGTCTGTGAA |
| 13 | octa_42nt_B1 | vertex | CCTTCATCAACCTCCGATTTTTTTGTGCCTTAACGCTCCTGCCTGAGGAG |
| 14 | octa_42nt_B2 | edge | CCAGCAGCAGTTGAGGACTTAACGTCGATT |
| 15 | octa_42nt_B3 | edge | ACGCTGCGATTTTTTTTTTTTTTTTTCCACACTTAACGAGGCCAACAAAAGTTGAACTCGTCGAATCCACCCTGCCTGC |
| 16 | octa_42nt_B4 | vertex | AAGATTCCGCAGCTCGCTTTTTTTAGACGGTCCTGTGGTTCTCCTGTTGAA |
| 17 | octa_42nt_B5 | edge | TACCCAAGAGTTGGGGGATTCATTGTGATG |
| 18 | octa_42nt_B6 | edge | CGGTGTTCTTTTTTTTTTTTTTTTTTTGGGAGTTTCTGTCTTCCTCCATAGAGGGCGGAACAGCTGATGGTGTACTTCCT |
| 19 | octa_42nt_B7 | vertex | TGCATTCTGTTTCTGGTTTTTTTAGAGAACTTTAGCATCTTCCATTGCTT |
| 20 | octa_42nt_B8 | vertex | GCGCGTAACAGGGCGCGTTTTTTTTCATATGGGGATTCCTTCAGGTTGTC |
| 21 | octa_42nt_B9 | edge | CCGCTACCACCACAGTGTAGCGGTCACGCT |
| 22 | octa_42nt_B10 | edge | CGGTGTTCTTTTTTTTTTTTTTTTTTAGAAAGCGAAAGGAGCGGGCGCTAGGGGCGAACGTGGCGAACGATGGTAATGCG |
| 23 | octa_42nt_B11 | vertex | CTTATAAATCAAATCATTTTTTTAGTTTTTTGGGGTCGAGGCTTATCGGC |
| 24 | octa_42nt_B12 | vertex | TCGCGGATGTTTCAGGTTTTTTTTCAGCAGCCTTGGCCGAAAGGAAGGGA |
| 25 | octa_42nt_C1 | edge | TCAGATCGTCACTTCAGTTCCCTGAGTGCT |
| 26 | octa_42nt_C2 | edge | CGGTGTTCTTTTTTTTTTTTTTTTTTGATCAGTGTGAACACAGCACCCACGAGTGCGGCTCTGGGACGACCAACGTGAGT |
| 27 | octa_42nt_C3 | vertex | ACAGAATGCATAGTATGTTTTTTTGTGAGTCCTTCAAGGACTCGAGATTGG |
| 28 | octa_42nt_C4 | edge | CAGTTTCTGGCGGTCAAAGCCTTTTCCTCCAGAGCTTGACAGTCATC |
| 29 | octa_42nt_C5 | vertex | CTTGACGGGAGCAGGTTTTTTTTCACCGAACAG |
| 30 | octa_42nt_C6 | vertex | ACTTCTCCCTTCCCAGATTTTTTTTGCTGACCTTGTCTGTACTTCAGGAT |
| 31 | octa_42nt_C7 | edge | CAGAGAAGCAGTAATGAGTTTCCTTCCAAGGATTCTTGAATGGCACA |
| 32 | octa_42nt_C8 | vertex | TGTGTTACCAGGATCTTTTTTTTGTGATGGGAA |
| 33 | octa_42nt_C9 | vertex | CCTGCGTGGCTGCAAATTTTTTTTATTCGTCCAGTTCGGGCCAGATCGTT |
| 34 | octa_42nt_C10 | edge | ATTGGTTGCGCGATTGTTGAGGTTGTAGTCACAGTGAGTTGCGAATG |
| 35 | octa_42nt_C11 | vertex | AGTGAGTGGGACTGAATTTTTTTTCACGGATGG |
| 36 | octa_42nt_C12 | vertex | ATGGGACATTTTCCAGTTTTTTTTCTTGCACAGTGCTGGTTGAGTGTTG |
| 37 | octa_42nt_D1 | edge | GTGGGCCACTTTTTTTTTTTTTTTTTTCCAGTTTGGAACAAGAGTCCACCCATAGCCCGAGATAGGCCACTACGTGA |
| 38 | octa_42nt_D2 | edge | ACCATCACCCAAAAGAGAAATCGGCAAAATCC |
| 39 | octa_42nt_D3 | vertex | GTGATCGGGTCCTCAATTTTTTTACAAGTAGGTCTTAGATGTTAGTATA |
| 40 | octa_42nt_D4 | edge | TCGCAGCGTTTTTTTTTTTTTTTTTCTGCTCCAATTTGTTCACCAGCTGCTTCGGAAGATGTGTCGTGATCTGTTCG |
| 41 | octa_42nt_D5 | edge | GCCAAGCACTGGGTCCCCAGAATACCGGACTC |
| 42 | octa_42nt_D6 | edge | AGAACACCGTTTTTTTTTTTTTTTTTGTTAGCCTCCTCAAACACCTGCTTCTTTGATCTGATTAGGATTTTAATCAGA |
| 43 | octa_42nt_D7 | edge | GCGTCAAATTGCTTTCGACACAGCTGTTCATA |
| 44 | octa_42nt_D8 | vertex | CAGCTGCTCAAACAGATTTTTTTCAGAGTTGATACCAATTTCCTTGGTA |
| 45 | octa_42nt_D9 | edge | GAGGTGAAGTGTAATGAGGTAACACTTGTC |
| 46 | octa_42nt_D10 | edge | ACGCTGCGATTTTTTTTTTTTTTTTATTTCCTGAACCTCTCAGGACGGTGACCGCTCTTGTTCAGTCATTGAAGATG |
| 47 | octa_42nt_D11 | edge | GCCACGAGCTGTTGTACAATGTGGCTCTCATT |
| 48 | octa_42nt_D12 | vertex | AGGGAAGGCATGATGCTTTTTTTTGCGGAAATCTTCAGCAACCCCAAGC |
| 49 | octa_42nt_E1 | edge | TCTGGTCCAGCCTGAGACGGCGCTCAGCAC |
| 50 | octa_42nt_E2 | vertex | AGATTCCTCTGCTCTTTTTTTTTTGGGCAGGGCTCCGGTCCTCATAGTC |
| 51 | octa_42nt_E3 | edge | CACCACCAGCCACTTCAGCTCATTGTCTGG |
| 52 | octa_42nt_E4 | edge | AACTCTCGAAAGCCAGCCCCCGATTTAGAG |
| 53 | octa_42nt_E5 | edge | TTCGAGGCTTCATGCGATGTCCTTCCAATC |
| 54 | octa_42nt_E6 | vertex | CTTGATCAGTAAACCCTTTTTTTACTTTCTCTGAATACTCGTTGAGGCT |
| 55 | octa_42nt_E7 | edge | GTGGCCCACTTTTTTTTTTTTTTTTAGGGATAACAATCTTATATTTGTCGCTGTTCTCCGCGGACTCGTGCCAATTC |
| 56 | octa_42nt_E8 | edge | ATCAGAAACTGACAGGGGAACAGATCCAGCCA |
| 57 | octa_42nt_E9 | edge | GTGGCCCACTTTTTTTTTTTTTTTTCAGCCAGGTTTCGACGTTATTGGATGTTCTTCATCTCCAGGCGTGCTTAGAC |
| 58 | octa_42nt_E10 | edge | TTCTCATCGCAGGGTCTCACGCACTCCTTCAG |
| 59 | octa_42nt_E11 | edge | GTGGCCCACTTTTTTTTTTTTTTTTGCGCTGGCATCTCTGCAATTGGTCTCTTCTCCTCCAGGAATGGTCCTTCTTG |
| 60 | octa_42nt_E12 | edge | ATATCTGTCGCTGCGCCGATGAAGTAGAAGCG |
| 61 | octa_42nt_F1 | edge | TCGCAGCGTTTTTTTTTTTTTTTTTTCTTCGTAGGTACCTATTAAAGAACGTATCAGGGCGGAATCCCTGCTGGAAC |
| 62 | octa_42nt_F2 | edge | AGAACACCGTTTTTTTTTTTTTTTTTGCT |
| 63 | octa_42nt_F3 | edge | GTGGGCCACTTTTTTTTTTTTTTTTACCACTCCCATCCTCAAAGCGGCGCTTACCAGGGACTGATGGGCCCACAATC |
| 64 | octa_42nt_F4 | edge | ACGCTGCGATTTTTTTTTTTTTTTTATAGCCTGTGTCAAGTCAACCATCTGACCTCAACTCTCCCAGGCCCATCTTC |
| 65 | octa_42nt_F5 | edge | GTGGGCCACTTTTTTTTTTTTTTTTCTCAGAGCCTGGACCTTGAGGGCTCTGGAGTTCTCTTGACATGGTATTCAGC |
| 66 | octa_42nt_F6 | edge | CGGTGTTCTTTTTTTTTTTTTTTTTTGTCTGGATCTGCATGATTCTTCCCATACACGAGGATCAATGCGTCCTGGATG |
| 67 | octa_42nt_F7 | edge | GTGGGCCACTTTTTTTTTTTTTTTTAGGATGACTTCGGCTCCTTTACCTGCAGTTCACCTTGAATCCCTCGGAATAG |
| 68 | octa_42nt_F8 | edge | TCGCAGCGTTTTTTTTTTTTTTTTTTCGGAACCCTAAAGGGGCGCTGGCAACCCGCCGCGTGCCGTAAAGCACTAAA |
| 69 | octa_42nt_F9 | edge | GTGGCCCACTTTTTTTTTTTTTTTTTCGCCCACCTGGTTGCCCCACAAATCCACCCTGGGGCAGGAGGCATCTATTG |
| 70 | octa_42nt_F10 | edge | TCGCAGCGTTTTTTTTTTTTTTTTTGGCAGACCCAGCAACACCTCAGATTGCTTTCCTGACCAGAGATGTGCCGCGA |
| 71 | octa_42nt_F11 | edge | AGAACACCGTTTTTTTTTTTTTTTTTGAGCTGCGTCCGGTCAAGGGCACCTTACTTCATTGGGTCCACAGCTCCCTGA |
| 72 | octa_42nt_F12 | edge | ACGCTGCGATTTTTTTTTTTTTTTTAGCAGGTCATCGTCCAGGATGGTCAGAGGTTGTGAACTGGCCCAAGATCTCC |
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 919
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 919
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 738
Illegal BglII site found at 1310 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 919
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 919
Illegal NgoMIV site found at 132 - 1000COMPATIBLE WITH RFC[1000]
References
Julin, S. et al., (2022). Dynamics of DNA Origami Lattices. Bioconjugate Chemistry, 34(1), 18-29. https://doi.org/10.1021/acs.bioconichem.2c00
Majikes, J.M. & Liddle, J.A. (January 8, 2021). DNA Origami Design: A How-To Tutorial. Journal of Research of the National Institute of Standards and Technology, 126:126001. https://doi.org/10.6028/jres.126.001
Nafisi, P. M. et al., (2018) Construction of a novel phagemid to produce custom DNA origami scaffolds, Synthetic Biology, Volume 3, Issue 1, https://doi.org/10.1093/synbio/ysy015
Noteborn, W. E. M. et al., (2020). One-Pot synthesis of Defined-Length SSDNA for multiscaffold DNA origami. Bioconjugate Chemistry, 32(1), 94–98. https://doi.org/10.1021/acs.bioconjchem.0c00644
Tandon, S. et al., (August 2021). Experimental investigation on tensile properties of the polymer and composite specimens printed in a Triangular pattern. Journal of manufacturing Process, 68A: 706-715. https://doi.org/10.1016/j.jmapro.2021.05.074
Wagenbauer, K. F. et al., (2017). How we make DNA origami. ChemBioChem, 18(19), 1873–1885. https://doi.org/10.1002/cbic.201700377
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