Coding

Part:BBa_K4829015

Designed by: Aditya Kamath Ammembal   Group: iGEM23_IISc-Bengaluru   (2023-10-05)


IVT of this sequence produces mRNA coding for a dAb against IL6

This composite biobrick is one of multiple combinations possible using our modular mRNA-based protein expression systems. We have not produced this using IVT. However, considering our success with BBa_K4829003, we can confidently say that this will produce the required dAb. We would like to emphasise further, that though this composite biobrick has used the CD33 signal peptide, we urge the users of the dAb for this application(mRNA protein production) to try out various different signal peptides!

Usage and Biology

  • Sequence coding for a dAb against IL6 (BBa_K4829009)

This composite biobrick is meant to be used in In Vitro Transcription/Translation. We would like to notify all users of this part that this DNA WILL NOT produce protein on transfection into mammalian cells.

  • We recommend that anyone using this construct use modified nucleotides: N1-Methylpseudouridine, and cap the mRNA with CleanCapAG, to improve translational efficiency and reduce immune response to the mRNA.
  • This specific dAb is expected to be a neutralising one. We have not used it for neutralising purposes ourselves yet. However, the potential use of this dAb would be to block IL6 either in disease processes or possibly even in cell biology assays/related studies.
  • It has a T7 promoter (BBa K4829000), a 5'UTR(BBa K4829004), a coding sequence (which has a signal peptide(BBa K4829001) along with the dAb(BBa_K4829009), a 3'UTR(BBa K4829005), and a polyA tail(BBa K4829006).
  • On In Vitro Transcription, followed by purification, the mRNA produced can be used to transfect HeLa cells with Lipofectamine messengerMax. After 6 hours, the protein will be detected in the supernatant.

Here are some potential uses of a dAb that binds to IL-6:

  • Therapeutic Applications:

Anti-inflammatory treatments: Given that IL-6 is involved in promoting inflammation, a dAb that neutralizes IL-6 could be used as a therapeutic agent to treat inflammatory conditions like multiple sclerosis, neuromyelitis optica spectrum disorder (NMOSD) or rheumatoid arthritis.

  • Cancer treatment: As detailed in Part:BBa_K4829008, IL-6 plays a vital role in TNBC progression through tumor growth promotion, angiogenesis, and stemness. Neutralizing IL-6 with a dAb might inhibit these processes and could be used as an effective therapy in triple-negative breast cancer treatment.
  • Treatment of infectious diseases: IL-6 plays a role in the pathogenesis of certain infections. Neutralizing IL-6 might modulate the host response, potentially reducing tissue damage and improving the outcome.
  • Diagnostic Applications:
  • Biosensors: dAb’s that bind IL-6 could be used in biosensors to detect and quantify IL-6 in biological samples, providing insights into inflammation or other pathological processes.
  • Imaging: Labeled dAb’s could be used in imaging modalities, like PET or MRI, to visualize sites of inflammation or tumors where IL-6 is overexpressed.
  • Research Tools:
    • Protein interaction studies: The dAb could be used to study the binding characteristics of IL-6 with its receptors or other interacting proteins.
    • Structural biology: Because of their small size and ability to bind specific epitopes, dAb’s can be excellent tools for crystallizing proteins or protein complexes for structural studies.
    • Cell-based assays: dAb’s could be used in cellular assays to study IL-6 signaling pathways or to neutralize IL-6 activity.
  • Drug Screening:

dAb’s can serve as a tool for high-throughput screening assays to identify compounds that interfere with IL-6 binding or function.

  • Drug Delivery:

Due to their small size and specific binding properties, dAb’s can be conjugated to drugs or other therapeutic agents and used to target them to specific sites where IL-6 is present.

  • Vaccine Development:

Although more exploratory, dAb’s could be used as a scaffold for vaccine development, especially if they can induce a neutralizing response against IL-6 or associated pathogens. We have not included his tag/FLAG tag in this sequence. However, it would be prudent to do so, for purification purposes. The specific features of this dAb have been modelled by our dry lab team, and the details may be found on our dry lab page. A brief summary of the details is mentioned below.

The image shown here is of the model of the dAb, predicted using Alphafold

  • il6nb.jpg
 Figure 1. The modelling of the dAb using alphafold2
  • il6nbdock.jpg 
 Figure 2. IL6(red) binding to our dAb(green)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 158
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters

The functional parameters we have been able to measure are the Gibbs free energy of binding and the Kd of the interaction with IL6. These details have been obtained using the GRAMM software. A full and detailed explanation of how this tool was used may be found on our dry lab page, and we encourage any users of this part to go through the extensive documentation there. We will post a few images of the binding parameters obtained below:

  • il6nbdata.jpg
 Figure 3. The binding parameters obtained from the GRAMM software
  • il6nbold.jpg
 Figure 4: The structure of the RNA pre-optimisation of the CDS by Ribotree. 'Loss' function value (AUP) = 139.12300000000005
  • scaleaup.jpg
 Scale of the AUP

For more information on the AUP, please check out out pages of BBa_K4829019 onwards

References

  • Browning L, Patel MR, Horvath EB, Tawara K, Jorcyk CL. IL-6 and ovarian cancer: inflammatory cytokines in promotion of metastasis. Cancer Manag Res. 2018 Dec 5;10:6685-6693. doi: 10.2147/CMAR.S179189. PMID: 30584363; PMCID: PMC6287645.
  • Zou, M., Zhang, X. & Xu, C. IL6-induced metastasis modulators p-STAT3, MMP-2 and MMP-9 are targets of 3,3′-diindolylmethane in ovarian cancer cells. Cell Oncol. 39, 47–57 (2016). https://doi.org/10.1007/s13402-015-0251-7
  • Joanna M. Watson, John L. Sensintaffar, Jonathan S. Berek, Otoniel Martínez-Maza; Constitutive Production of Interleukin 6 by Ovarian Cancer Cell Lines and by Primary Ovarian Tumor Cultures1. Cancer Res 1 November 1990; 50 (21): 6959–6965.
  • Patricia E. Ellis, Gemma A. Barron, Giovanna Bermano,Adipocytokines and their relationship to endometrial cancer risk: A systematic review and meta-analysis,Gynecologic Oncology,Volume 158, Issue 2,2020,Pages 507-516,ISSN 0090-8258, https://doi.org/10.1016/j.ygyno.2020.05.033.
  • Christine M. Friedenreich, Annie R. Langley, Thomas P. Speidel, David C.W. Lau,Kerry S. Courneya, Ilona Csizmadi, Anthony M. Magliocco, Yutaka Yasui and Linda S. Cook. Case–control study of inflammatory markers and the risk of endometrial cancer. European Journal of Cancer Prevention , JULY 2013, Vol. 22, No. 4 (JULY 2013),pp. 374-379. https://www.jstor.org/stable/10.2307/48504258
  • Shuwei Liang, Zhuojia Chen, Guanmin Jiang, Yan Zhou, Qiao Liu, Qiao Su, Weidong Wei, Jun Du, Hongsheng Wang, Activation of GPER suppresses migration and angiogenesis of triple negative breast cancer via inhibition of NF-κB/IL-6 signals, Cancer Letters,Volume 386,2017,Pages 12-23,ISSN 0304-3835,https://doi.org/10.1016/j.canlet.2016.11.003.
  • Tian, J., Chen, X., Fu, S. et al. Bazedoxifene is a novel IL-6/GP130 inhibitor for treating triple-negative breast cancer. Breast Cancer Res Treat 175, 553–566 (2019). https://doi.org/10.1007/s10549-019-05183-2
  • Zachary C. Hartman, Graham M. Poage, Petra den Hollander, Anna Tsimelzon, Jamal Hill, Nattapon Panupinthu, Yun Zhang, Abhijit Mazumdar, Susan G. Hilsenbeck, Gordon B. Mills, Powel H. Brown; Growth of Triple-Negative Breast Cancer Cells Relies upon Coordinate Autocrine Expression of the Proinflammatory Cytokines IL-6 and IL-8. Cancer Res 1 June 2013; 73 (11): 3470–3480. https://doi.org/10.1158/0008-5472.CAN-12-4524-T
  • Jin, K., Pandey, N.B. & Popel, A.S. Simultaneous blockade of IL-6 and CCL5 signaling for synergistic inhibition of triple-negative breast cancer growth and metastasis.
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