Fig. 4: Stabilization of p53 and activation of apoptosis via LNPs encapsulating p53-targeting duAbs.

A Programmable design of p53-targeting duAbs, encapsulation and delivery via mRNA-encapsulated lipid nanoparticles (LNPs), and downstream apoptosis activation. Created in BioRender. Wang, T. (2025) https://BioRender.com/x61p552. B Stabilization of endogenous p53 in protein extracts of HeLa cells analyzed by immunoblotting. HeLa cells were transiently transfected with a pcDNA3 plasmid encoding one of the candidate duAbs while transfection with a polyG peptide-guided duAb served as a control. An equivalent amount of protein was loaded in each lane. Blots were probed with anti-p53 and anti-GAPDH antibodies and are representative of biological replicates (n = 3). C Stabilization of endogenous p53 in protein extracts of HeLa cells after the best p53-stabilizing duAb (p53_pMLM_4-OTUB1) was delivered via LNPs analyzed by immunoblotting. HeLa cells were transiently transfected with LNPs encapsulating p53_pMLM_4-duAbs encoded as mRNA (loaded 1 μg and 2 μg, respectively) while transfection with luciferase-encoding mRNA-LNP served as a control. An equivalent amount of protein was loaded in each lane. Blots were probed with anti-p53 and anti-Vinculin antibodies and are representative of biological replicates (n = 3). D Increase in apoptosis hallmark cleaved-PARP-1 (Cl-PARP-1) in protein extracts of HeLa cells after the best p53-stabilizing duAb (p53_pMLM_4-OTUB1) was delivered via LNPs analyzed by immunoblotting. HeLa cells were transiently transfected with LNPs encapsulating p53_pMLM_4-duAbs encoded as mRNA (loaded 1 μg and 2 μg, respectively) while transfection with luciferase-encoding mRNA-LNP served as a control. An equivalent amount of protein was loaded in each lane. Blots were probed with anti-Cl-PARP-1 and anti-Vinculin antibodies and are representative of biological replicates (n = 3). For all immunoblots in (B–D), an equivalent amount of protein was loaded in each lane. Molecular weight (MW) ladder is indicated at left. Intensity of target protein bands was calculated via densitometry and normalized to intensity of GAPDH and Vinculin loading controls and then normalized to applicable controls (polyG-OTUB1 for (B) and luciferase LNP for (C) and (D)). Data are the average of biological replicates and technical replicates (n = 3 for p53 and Cl-PARP-1). Statistical analysis for this figure was performed using the two-tailed Student’s t-test using GraphPad Prism 10 software, with calculated p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The p values above each bar in the fold stabilization and densitometry analyses represent the comparison between the control (polyG-OTUB1, luciferase LNP) and the respective sample; all other p value notations compare the specified samples. Please refer to source data for numeric p values. The structure for p53 was predicted via the AlphaFold3 server, and the shading was done according to AlphaFold’s confidence metric, plDDT, as follows: very low (plDDT <50) = orange, low (70 > plDDT > 50) = yellow, confident (70 > plDDT > 90) = light blue, very high (plDDT > 90) = light blue.