Near-Infrared Luciferase Complementation Assay with Enhanced Bioluminescence for Studying Protein–Protein Interactions and Drug Evaluation Under Physiological Conditions

Identification of protein–protein interactions (PPIs) that occur in various cellular processes helps to reveal their potential molecular mechanisms, and there is still an urgent need to develop the assays to explore PPIs in living subjects. Here, we reported a near-infrared split luciferase complementation assay (SLCA) with enhanced bioluminescence produced by cleaving a luciferase, Akaluc, for exploring and visualizing PPIs in living cells and live mice. Compared with the previously developed and widely used red SLCA based on split firefly luciferase (Fluc-SLCA), the signal intensities for PPI recognition in living cells and live mice of the Akaluc-SLCA increased by ∼3.79-fold and ∼18.06-fold in the measured condition, respectively. Additionally, the interactions between the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular RNA processing proteins were identified, and the drug evaluation assays were also performed in living cells using Akaluc-SLCA. This study provides a new tool in the near-infrared region for the identification of PPIs in living cells and in vivo and new information for the understanding and treatment of SARS-CoV-2

hiv1-miR-N367 and hsa-miR192 act as functional orthologs.

<p>(A) Sequence homology between hiv1-miR-N367 and hsa-miR192 (marked in red). The seed regions of miRNAs were underlined. (B) Sequences of miR192 and its non-fully complementary target. (C) Fluorescence reporter assay of the indicator vectors pmiR-N367:4tar(n), pmiR192:4tar(n), pmiR-N367:4tar(192), pmiR192:4tar(192) and their control vectors with miRNAs or target sequences only. (D) Fluorescence reporter assay of the indicator vectors pmiR-N367:PABPC4UTR, pmiR192:PABPC4UTR and their control vectors with miRNAs only, with miRNA deletions or without miRNA pairing sites in the 3′-UTR.</p

Validation of the dual-fluorescent protein reporter system as an miRNA functional assay.

<p>(A) Sequences of miR30, hiv1-miR-N367 and their non-fully complementary targets. (B) Northern blot analysis of the transcription of specific miRNAs. HeLa cells were mock-transfected (mock) or transfected with plasmids pmiR30, pmiR-N367 individually, and the location of the mature miR30 and miR-N367 is indicated. tRNA^Val served as a loading control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036157#pone.0036157-Tran1" target="_blank">[26]</a>. (C) Fluorescence reporter assay of the indicator vectors pmiR30:4tar(30), pmiR-N367:4tar(n) and their control vectors. HeLa cell cultures were transfected with the indicator vectors pmiR30:4tar(30), pmiR-N367:4tar(n) and their control vectors with miRNAs or target sequences only. The ratio of EGFP to mCherry fluorescence intensity is shown.</p

sv40-miR-S1-5p and hsa-miR423-5p downregulate the predicted biological targets of hsa-miR423-5p.

<p>HeLa cultures were transfected by the indicator vectors pmiR-S1-5p:DMWDUTR, pmiR423-5p:DMWDUTR, pmiR-S1-5p:C20orf27UTR, pmiR423-5p:C20orf27UTR and their control vectors with miRNAs only, with miRNA deletions or without miRNA pairing sites in the 3′-UTR.</p

Monitoring miRNA activities in living cells.

<p>Activities of two miRNAs, including miR30 (A), miR-N367 (B), were imaged in live HeLa cell cultures that were transfected with the indicator vectors pmiR30:4tar(30), pmiR-N367:4tar(n) and their control vectors with miRNAs or target sequences only, respectively. Bar, 10 μm.</p

The construction and principle behind the dual-fluorescent protein reporter vector.

<p>(A) A diagram of the dual-fluorescent protein reporter vector pMGhU6. (B) The principle of the dual-fluorescent protein reporter vector system.</p

Model for the iBRB breakdown affected by EBOV.

EBOV stimulates pericytes to secrete VEGF to cause the iBRB breakdown. VEGF disrupts iBRB through downregulation of the tight junction protein claudin-1. The VEGF antibody Avastin can reduce the effect on VLP-induced iBRB breakdown.</p

EBO-VLPs downregulate the tight junction protein Claudin-1 in the iBRB.

(A) Western blot analysis of claudin-1, occludin and ZO-1 expression in HREC at 48 h in the tri-culture iBRB model. Representative images of three independent experiments are shown. (B) Relative protein levels of claudin-1, occludin and ZO-1 were normalized to β-actin. The results are presented as the means ± standard deviation of three independent experiments. (C) Immunofluorescence images of claudin-1, occludin and ZO-1 expression in HRECs at 48 h post EBO-VLP treatment in the tri-culture iBRB model. Representative images of three independent experiments are shown. (D) Quantification of the fluorescence intensity of claudin-1, occludin and ZO-1 after EBO-VLP administration. The regions for fluorescence intensity analysis were determined in four independent experiments. **p < 0.01, ***p < 0.001.</p

EBO-VLPs can significantly stimulate pericytes to secrete VEGF.

(A) Representative cytokines secreted by HREC, HRP and HRA after 48 h of EBO-VLP stimulation. Representative images of three independent experiments are shown. (B) Relative cytokine levels were normalized to the mock group without EBO-VLP administration, which was set as 1. The floating bar plot shows the mean, minimum and maximum levels of each cytokine in three independent experiments. Downregulation of cytokines expression were displayed with negative numbers. (C) Changes in VEGF expression after EBO-VLP administration, as quantified by ELISA. The horizontal dashed line marks the limit of detection of the assay. The results are presented as the means ± standard deviation of three independent experiments. Statistical analysis was performed using Student’s t test. *p < 0.05, **p < 0.01.</p

Characterization of iBRB co-culture models by HRECs and HRAs.

Related to Fig 4. (A) Assessment of integrity of co-culture iBRB in vitro barrier models with HRECs and HRAs by TEER every day in a week. The results are presented as the means ± standard deviation of six independent experiments. (B) Na-F permeability of iBRB co-culture models at 6 hours and 3 days post HREC seeding. The results are presented as the means ± standard deviation of four independent experiments. Statistical analysis was performed using Student’s t-test. (C) Images of HRECs showing expression of claudin-1, occludin, ZO-1 in co-culture with HRECs and HRAs. Claudin-1, occludin, ZO-1 are shown in green and cell nuclei stained with DAPI (blue). Representative images of three independent experiments are shown. The fluorescent Images were taken at 60× magnification objective lens under a confocal microscope. Statistical analysis was performed using Student’s t-test. ***p (TIF)</p