Hypothesis

Light‑activated modulation of RNA‑binding protein (RBP) affinity for EXOmotifs can be used to selectively increase RNA cargo loading in hybrid exosomes, enabling spatiotemporally controlled therapeutic release.

Mechanistic Rationale

Hybrid exosomes combine the natural membrane of exosomes with the high‑capacity lipid bilayer of liposomes, giving them superior drug loading and low immunogenicity (Hybrid exosomes achieve superior drug loading and targeting). Natural exosomes sort RNAs via RBPs such as YBX1, hnRNPA1 and hnRNPC1 that bind short sequence motifs (EXOmotifs) like CAUUG or UCAGU during intraluminal vesicle formation (EXOmotif‑RBP interactions enable controlled miRNA sorting). Recent work shows that photoactivatable synthetic exosomes can trigger click‑chemistry–mediated surface modifications upon light exposure (photoactivatable exosomes provide controlled delivery). We hypothesize that incorporating a light‑sensitive domain (e.g., LOV2) into the RBP YBX1 will alter its RNA‑binding affinity in a wavelength‑dependent manner. In the dark state the LOV2 domain sterically hinders the RBP’s RNA‑recognition motif, reducing EXOmotif binding; blue‑light illumination induces a conformational change that exposes the RNA‑binding surface, increasing affinity for target motifs and thereby boosting sorting of the corresponding RNA into the intraluminal vesicle of the hybrid exosome.

Experimental Design

1. Construct engineering – Fuse a LOV2 photoreceptor to the N‑terminus of YBX1 via a flexible linker; generate a control YBX1‑LOV2 mutant where the LOV2 domain is locked in the open state.
2. Cell‑based production – Transfect HEK293 cells expressing either wild‑type YBX1, YBX1‑LOV2, or the locked‑open mutant together with a reporter RNA containing multiple CAUUG motifs fused to a fluorescent protein (e.g., GFP‑CAUUGx4). Produce hybrid exosomes by co‑transfecting a liposome‑fusion plasmid (e.g., VSV‑G‑linked phosphatidylserine) to generate exosomes‑liposome hybrids.
3. Light treatment – Split harvested hybrid exosomes into dark‑kept and blue‑light (470 nm, 5 mW cm⁻², 5 min) groups.
4. Cargo quantification – Isolate exosomes via size‑exclusion chromatography, extract RNA, and measure reporter RNA levels by RT‑qPCR and GFP fluorescence. Perform parallel western blot for YBX1‑LOV2 expression.
5. Specificity test – Conduct CLIP‑seq on YBX1‑LOV2 from dark and light conditions to confirm light‑dependent increase in CAUUG binding peaks.
6. Functional readout – Deliver the hybrid exosomes to recipient cells lacking the reporter and assess GFP expression after light exposure versus dark.

Expected Outcomes

• Dark‑treated hybrid exosomes will show baseline reporter RNA loading comparable to wild‑type YBX1.
• Blue‑light‑treated YBX1‑LOV2 hybrids will exhibit a ≥2‑fold increase in reporter RNA incorporation relative to dark controls.
• Locked‑open YBX1‑LOV2 will display constitutively high loading irrespective of light, confirming that the effect is due to the photoswitchable domain.
• CLIP‑seq will reveal enriched CAUUG peaks only in the light‑treated samples.
• Recipient cells receiving light‑activated hybrids will show higher GFP expression, demonstrating functional cargo transfer and light‑controlled release.

Potential Pitfalls and Alternatives

• If LOV2 fusion impairs YBX1 stability, we can test alternative photoswitchable domains (e.g., PhyB‑PIF) or use chemogenetic dimerizers.
• Exosome heterogeneity may obscure cargo measurements; we will normalize to exosome particle count (NTA) and CD63 western blot.
• Light penetration in vivo is limited; for deep‑tissue applications we could shift to red‑shifted photoreceptors (e.g., BphP) or use upconversion nanoparticles to convert NIR light to visible.

This hypothesis directly links the mechanistic insight of RBP‑motif recognition with the engineered hybrid exosome platform, offering a testable route to achieve precision, on‑demand RNA therapeutics.