STRC Research

STRC In Situ SpyCatcher Assembly

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STRC In Situ SpyCatcher Assembly

Two synthetic protein fragments, each bearing one half of a split-SpyCatcher / SpyTag pair, diffuse into endolymph and covalently assemble in situ into a functional STRC-equivalent scaffold at the stereocilia–tectorial-membrane interface. No cell is ever transfected. No ribosome is ever involved.

The hidden assumption this breaks

Earth gene therapy assumes the cell must make the protein. But STRC is extracellular — it leaves the cell to work. If the cell never needs to see it, the whole gene-delivery stack (AAV, LNP, mRNA, capsid immunity, dual-vector recombination) is unnecessary overhead.

Mechanism

SpyCatcher/SpyTag system (Howarth lab, 2012; now FDA-precedent in vaccine scaffolds): a 138-residue domain and a 13-residue peptide form an isopeptide bond spontaneously on contact. Irreversible, covalent, physiological pH, no enzymes.

Construct design:

  • Fragment A: actin-binding peptide (~30 aa) — SpyTag (13 aa) — flexible linker (20 aa) — small soluble globule (~50 aa) = ~113 aa total
  • Fragment B: TMEM145-binding peptide (C-terminal ARM-repeat mimic, ~80 aa) — SpyCatcher (138 aa) — flexible linker (20 aa) = ~238 aa total

Each fragment produced separately in E. coli or cell-free system; purified; formulated as co-injectable solution. Delivered via round-window injection or, with sonoporation assist, as an ear drop.

Upon injection the two fragments diffuse independently through perilymph. When they meet at a stereocilia tip (actin-enriched) or at a TM-proximal site (TMEM145-enriched), they encounter each other’s tag/catcher at high local concentration and form the SpyBond. Result: a covalent synthetic heterodimer mechanically linking stereocilium actin to TM-bound TMEM145.

Why this works where soluble STRC might not

  • Covalent assembly is irreversible — thermodynamically dominant over reversible non-covalent interactions. Even at low fragment concentration, once bonded the construct stays bonded.
  • Spatial targeting is free: Fragment A localizes to actin, Fragment B to TMEM145 — they only finish forming where both partners exist. This is nanoscale addressable self-assembly.
  • Each fragment is small (<250 aa) — trivially producible in commercial E. coli at 1000/mg.

Computational proof path

  1. AF3-Multimer of Fragment A + actin and Fragment B + TMEM145 GOLD separately — confirm binding interfaces before assembly. Target ipTM > 0.6 each.
  2. AF3-Multimer of assembled A-SpyBond-B — confirm the joined construct folds sensibly; check that the linker lets A and B adopt compatible orientations. (SpyCatcher/SpyTag geometry is well-characterized: exit vector ~15 Å.)
  3. Diffusion-reaction ODE — model [A], [B], [A·B] in perilymph over time with second-order bimolecular rate (SpyTag/SpyCatcher k_on ≈ 10³ M⁻¹s⁻¹, published). Output: fraction of stereocilia decorated vs injected dose.
  4. Mass-balance / therapeutic-dose estimate — OHCs = ~12,000 per ear × ~100 HTC-equivalent sites per cell = 1.2 × 10⁶ assembled constructs needed. Perilymph volume = ~50 µL. Required fragment concentration ≈ 50 nM. Injectable. Straightforward.
  5. Bundle stiffness after assembly — plug into STRC Stereocilia Bundle Mechanics Model, compute f-equivalent.

Why no one has tried this in the cochlea

SpyCatcher is the domain of vaccine scaffolding and biotech research reagents — used to decorate virus-like particles with antigen arrays. No group combines split-protein covalent chemistry with cochlear therapeutics. It is a cross-field orphan.

Risks

  • Immune response to SpyCatcher (Streptococcus pyogenes origin) — moderate, but intracochlear compartment is immune-privileged early in life.
  • Mis-assembly in perilymph bulk (Fragment A + Fragment B meet far from stereocilia) — mitigated by short linker + low concentration + kinetic preference for surface-enriched partners.
  • Covalent bond is permanent — no off switch. Acceptable for structural replacement (same as gene therapy).

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