To complement the self-closing mechanism of our spring-loaded box, a fusion protein has been designed and produced to function as the trigger and courier for a potential therapeutic cargo. Experiments were conducted to characterise the functionality of the trigger, its activity after cargo conjugation and its interaction with DNA origami structures. Results support that we have developed a recombinant protein which:
- cleaves double stranded DNA with high sequence recognition specificity
- retains enzymatic functions upon conjugation with small peptides
- displays potential for a time-controlled release mechanism once trapped inside our spring-loaded origami boxes
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To produce a payload model which triggers the capturing mechanism of our box, we designed a fusion protein with several characteristics. The core functions which were desired included:
- The ability to cut the double stranded brace on the box to trigger the closing mechanism
- The versatility of carrying a wide range of therapeutic biomolecular cargoes into the box
- The possibility of providing ease in quantifying the success of payload capture
The above objectives were met through designing a fusion protein consisting of a cysteine tagged endonuclease attached to a SpyCatcher protein through a flexible linker (Gly-Gly-Ser-Gly). Upon contact with our origami boxes, the endonuclease should recognise the specific sequence encoded on the double stranded brace and perform cleavage of the DNA. This would trigger the spring loaded mechanism of the box and capture the payload. To maximise chances of payload encapsulation, the polyhistidine tag inherent on our recombinant trigger molecule will be leveraged to perform reversible binding to a Ni-NTA staple within the cavity of our box. Attachment of the trigger to the box can be counteracted through applying imidazole, a common technique in Nickel affinity chromatography. In addition, as the endonuclease is engineered with a SpyCatcher construct, a diverse range of biomolecules may be chemically conjugated to the enzyme.
Endonucleases, also known as restriction enzymes, are of bacterial origin and function as a defence mechanism which cleaves foreign dsDNA upon recognition of a specific sequence (Goodsell, 2002). In nature, the host bacteria are immune to the detrimental effects of this enzyme through methylation of the recognised cut sites. DNA methylation is catalysed by modification methylases, which have co-evolved with corresponding endonucleases (McClean, 1998).
For this project, we aimed to select an endonuclease which was not capable of damaging the M13-based origami box while being able to cleave at a unique recognition sequence on the brace. After identifying the non-cutters of the M13 sequence, we nominated EcoRI as our core enzyme.
We anticipated challenges in producing this enzyme as it has the potential to destroy the genome of the host cell during expression. To mitigate this risk, we decided to co-transform expression hosts with methylase genes to protect genomic DNA.
The SpyCatcher is a recombinant protein, originating from Streptococcus pyogenes fibronectin-binding protein (FbaB) (Kang, et al., 2007), which was found to chemically conjugate to a short peptide, SpyTag, through isopeptide bonds (SC. & Howarth, 2015). Further optimisation of the proteins was carried out to truncate the SpyCatcher molecule without compromising functionality (Li, et al., 2014). The SpyCatcher-SpyTag complex is often used for its adhesive properties where separate biomolecular entities can be connected via this construct. Through this interaction, we are able to biologically conjugate almost any protein or peptide cargo to form a part of our payload.
|Conjugated Structure||Peptide||Small biomolecule||Cargo molecule of our payload||Potentially deliver therapeutic components|
|SpyTag||Peptide which adheres to SpyCatcher||Part of adaptor structure between the endonuclease and any DNA or protein||Attach endonuclease to DNA box or therapeutic proteins|
|Fusion Protein||Polyhistidine Tag||Peptide tag which assists Ni-Affinity Chromatography||Typically used for protein purification. Leveraged as mechanism for reversible binding to box.||To ensure encapsulation of the trigger upon closing of box|
|SpyCatcher||Protein which adheres to SpyTag||Part of adaptor structure between the endonuclease and any DNA or protein||Attach endonuclease to DNA box or therapeutic proteins|
|Endonuclease||Enzyme which cleaves dsDNA||Cleave dsDNA brace||Trigger closing mechanism|
|Cysteine||Accessible residue at the C-terminus of protein||Site for maleimide fluorophore dye||Allow detection and validation of protein|
Table 1: Description and function of different components of the payload
In order to program E. Coli to produce specific proteins, the DNA sequence encoding the desired product is delivered into the cell to generate a clone with the same genetic lineage. To transfer genetic material into bacteria for protein production, a suitable expression vector must be used. For our project, we decided to clone using Sequence and Ligation Independent Cloning vectors (SLIC), which are a set of 8 pACYC-Duet1 plasmids that differ by N- and C-terminus tags (see Appendix for details on SLIC A-H vectors). In addition to short peptide tags which bind to Ni-NTA beads to assist the process of protein purification, the plasmids contain antibiotic resistance markers for selection, T7 promoter regions that control the expression of proteins and a multiple cloning site for the insertion of the DNA sequence encoding the desired product.
Insertion of the desired DNA sequence into SLIC vectors, linearised by EcoRI digestion, involves the generation of single stranded complementary sticky ends on both constructs. Through complementary base pairing, the inserts join up with the vectors and re-circularise. These plasmids are then transformed into high copy number E. coli to produce replicates of the genetic material. Once harvested and confirmed, the recombinant DNA is delivered into expression cells for the production of proteins.
Production of recombinant proteins is carried out by bacterial cells containing genetic sequences encoding for the desired product. Following production, purification processes are performed by extracting the His-tagged protein through its affinity for Ni-NTA (Bornhorst & Falke, 2000). As the particles selectively bind to 10xHistags, which are encoded for on the SLIC vectors, it is expected that after elution, the remaining solution would contain recombinant product. Samples are then characterised using a combination of techniques including SDS-Polyacrylamide gel electrophoresis, Western Blotting, mass spectrophotometry and enzyme assays (Lodish, et al., 2000).
To clone and purify a recombinant SpyCatcher-endonuclease protein to complement the function of our self-closing DNA origami box.
SLIC and Restriction Enzyme Cloning
Figure 1: cPCR samples loaded on 1% agarose gel demonstrating the presence of SpyCatcher-EcoRI inserts in SLIC vectors.
Figure 2: cPCR samples loaded on 1% agarose gel demonstrating the presence of EcoRI methylase inserts in pET-Duet1 vectors.
To engineering bacteria to produce recombinant proteins, genetic sequences encoding for the product must be cloned into an expression plasmid vector. The success of cloning attempts can be verified by cPCR which involves amplification of the multi-cloning site of the plasmid. cPCR samples can indicate whether the DNA insert has been integrated to the plasmid. If DNA bands of the control and variables show a size difference similar to the expected insert length, cloning has been achieved. It can be clearly seen in Figure 1 that the amplified regions in lane 10 to 15 are longer than the empty control plasmid in lane 9. Similar trends are identified for lanes 4 to 7 in Figure 2, suggesting the presence of the DNA insert within the vector. Our fusion protein was produced from the colony shown in lane 15 of Figure 1 while the methylase was obtained from the lineage characterised in lane 4 of Figure 2.
His-tag Protein Purification
Figure 3: Western blot confirming presence of purified SpyCatcher-EcoRI.
To purify our protein from cell lysate, the polyhistidine tag on the trigger was utilised to perform nickel affinity chromatography. Several stages of the process were sampled to track potential loss of protein products. Typically, SDS-PAGE gel electrophoresis is used to confirm the production of recombinant proteins. However, due to the toxicity of our trigger towards the expression host, concentrations of the molecule were beyond detection sensitivity and Western Blots were carried out to identify the presence of the protein. Western blot results reveal that the elution sample of our double transformants (SpyCatcher-EcoRI and M.EcoRI) contained protein products with affinity for His-tag antibodies. As the bands were unique to the double transformants, it can be confirmed that it was not a false positive result.
Endonuclease Activity of SpyCatcher-EcoRI
Figure 4: Digestion of brace with opening angle of 135 degrees and dsDNA without EcoRI recognition sequence loaded on 10% polyacrylamide gel.
Enzyme assays were performed with our trigger molecules to confirm its behaviour. Double stranded DNA with and without the EcoRI recognition site were incubated with the SpyCatcher-EcoRI to test for activity and specificity. Gel electrophoresis was conducted to examine whether the DNA molecules were cleaved. Accurate sequence recognition by the SpyCatcher-EcoRI trigger is demonstrated by the identical sizes of the two smaller DNA bands in both our experimental and commercial digests. Digestion of our double stranded 135 degree brace confirms compatibility of the trigger with our dynamic origami box structure. In addition, non-specific cutting can be ruled out since no digestion of the dsDNA, which lacks an EcoRI recognition site, is observed. As heat inactivation is used to halt enzyme activity, the dsDNA samples were unhybridised and two bands are shown in the gel.
Peptide to SpyCatcher Conjugation
Figure 5: Digestion of 135⁰ dsDNA brace with FastDigest EcoRI and the SpyCatcher-EcoRI trigger with or without peptide conjugation. Samples were run on 10% polyacrylamide gel.
To confirm that the trigger is still active after conjugation of additional biomolecules via the SpyCatcher region, an enzyme assay was conducted on SpyTag bound proteins. Identical digestion bands across the three lanes in figure 5 show that peptide-conjugated SpyCatcher-EcoRI triggers have the same cleaving behaviours as commercial and non-conjugated triggers. These results suggest that our recombinant enzyme is capable of acting as a transport vector to carry small cargo into the spring-loaded DNA box for packing without compromising endonuclease activity.
Enzyme effect on DNA origami
Figure 6: Time series digestion of DNA Origami structures with FastDigest EcoRI and SpyCatcher-EcoRI.
The interaction of the trigger molecule with DNA origami structures were examined by performing digestion reactions. Samples of our box and female origami barrels incubated with the enzymes for various lengths of time were electrophoresed on a TBE agarose gel to observe characteristics of star activity. The digestion trends of DNA origami shown in Figure 6 suggest potency of both the FastDigest EcoRI and SpyCatcher-EcoRI triggers in cleaving the structures. As incubation times increase, more of the origami becomes degraded by enzyme catalysis. This reflects the possibility of implementing a time controlled release mechanism for our payload once it is trapped into the spring-loaded box and delivered to the desired destination.
Materials and Methods
Cloning of the fusion protein
GBlocks for our synthetic endonucleases (SpyCatcher-EcoRI, SpyCatcher-SpeI and SpyCatcher-XbaI) were ordered from IDT to be compatible with Sequence and Ligation Independent Cloning vectors (SLIC). Once inserts were ligated into SLIC vectors, transformation of these products were carried out on NEB DH5α competent E. coli (High efficiency), which were screened using colony PCR. Successful cloning attempts were confirmed by a combination of colony PCR, confirmatory digestion and Sanger Sequencing. Identified colonies were amplified by 20mL grow ups in LB broth and plasmid samples were obtained using Minipreps (Qiagen) Plasmid Purification.
To account for the possibility of the endonucleases in inducing death of the expression chassis by cleavage of genomic DNA, we cloned corresponding methylases using traditional restriction enzyme methods. To protect DNA of the expression host, we anticipated to produce double transformants which contained methylases capable of methylating recognition sites to prevent endonuclease activity. Sequences were ordered as gBlocks and ligated to pET-Duet1 vectors via NcoI and EcoRI cut sites. The upstream restriction enzyme sequence was specifically chosen to bypass the His-tag coding on the plasmid, in order to prevent extraction of methylase proteins during the purification of our desired recombinant enzyme. All subsequent procedures were identical to cloning of the fusion protein.
Purified SpyCatcher-endonuclease and methylase plasmid pairs were used to transform NEB T7 expression cells which each experimental set containing single transformants with a SpyCatcher-endonuclease or a methylase and double transformants. Bacteria were grown in LB media at 37 degrees, induced at OD 0.5 and incubated overnight at 18°C. The target protein was purified using Nickel Affinity Chromatography and samples retrieved from various stages were examined on SDS-PAGE. As our product concentration was below the detection threshold of gel electrophoresis, a confirmatory Western Blot was carried out. Once confirmed, elution samples were concentrated and buffer exchanged using Spin Columns.
Recombinant enzyme assay
Time and dilution series of DNA digestion with our purified SpyCatcher-EcoRI was performed to investigate the optimal experimental conditions. pRSF-Duet1 vectors were incubated with the enzymes for varied lengths of time and samples were examined using gel electrophoresis. Results were compared with uncut and commercially cut plasmid to assess activity levels.
For further confirmation, incubation was carried out with SpyCatcher-EcoRI and our opening brace of 135 degrees (see DNA design) and samples were ran on 10% acrylamide gels. To confirm that our protein does not cut non-specifically, assays were performed on dsDNA that does not contain the EcoRI recognition site.
Peptide to SpyCatcher Conjugation
To confirm that the recombinant enzymes retain its DNA cutting functions upon the addition of biomolecules to the SpyCatcher region, SpyTag peptides were conjugated onto the protein. Enzyme assays were then performed to assess whether the peptides impact on the activity of the protein.
Enzyme effect on DNA origami
To assess the impact of SpyCatcher-EcoRI on DNA origami structures, incubations of the enzyme with different constructs were carried out for varied amount of time. Samples were run on agarose gel, alongside commercial digests, to detect whether non-specific cleaving of structures has occurred.
We have successfully clone and purified active recombinant endonuclease coupled with a SpyCatcher protein. While less efficient and reveal higher star activity than the commercial FastDigest EcoRI, results show that with controlled incubation time and concentration, our fusion enzyme has directed DNase activity. It is also shown that addition of biomolecules at the N-terminus of the protein via chemical conjugations with the SpyCatcher does not affect the activity of the endonuclease, suggesting a range of possibilities in targeted delivery.
Due to time constraints, our endeavours in thoroughly validating and characterising the mechanisms of the trigger and its interaction with our origami box could not be carried forward. While results suggest that the SpyTag-SpyCatcher conjugation does not affect the activity of the endonuclease on the fusion protein, further experiments to examine attachment of larger cargo would be preferred. It would provide powerful validation to our project if we could quantify capturing efficiency of bound and unbound payload through TEM and single molecule FRET.
SLIC Vector Tags
|N-terminus Tag||C-terminus Tag|
DNA Sequences can be found here.
Bornhorst, J. A. & Falke, J. J., 2000. Purification of Proteins Using Polyhistidine Affinity Tags. Methods Enzymology, Volume 326, pp. 245-254.
Goodsell, D., 2002. The Molecular Perspective: Restriction Endonucleases. The Oncologist, 7(1), pp. 82-83.
Kang, H. et al., 2007. Stabilizing isopeptide bonds revealed in gram-positive bacterial. Science, Volume 318, pp. 1625-1628.
Li, L., Fierer, J., Rapoport, T. & Howarth, M., 2014. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. Journal of Molecular Biology, 426(2), pp. 309-317.
Lodish, H., Berk, A. & Zipursky, S., 2000. Section 3.5 Purifying, Detecting, and Characterizing Proteins. In: Molecular Cell Biology 4th Edition. New York: W.H. Freeman.
McClean, P., 1998. Restriction Modification System. [Online] Available at: https://www.ndsu.edu/pubweb/~mcclean/plsc731/dna/dna5.htm [Accessed 18 10 2016].
SC., R. & Howarth, M., 2015. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Current Opinion in Chemical Biology, Volume 29, pp. 94-99.