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Geoffrey Weiner, Shweta Sharma, Tarmo Mulder, Sahil Shah, Christian Angelopoulos, Erkki Ruoslahti, Jeffrey Goldberg; In vivo phage display uncovers retinal endothelium-homing motifs in normal and optic nerve crushed eyes.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3619.
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© ARVO (1962-2015); The Authors (2016-present)
To determine, using in vivo phage display, whether specific homing peptides can be identified for retinal endothelium in both normal and optic nerve crushed (ONC) eyes. We hypothesize that retinal ganglion cell health influences the expression of surface proteins on local endothelial cells, and here we assay for differential expression by in vivo phage display of a small cyclic peptide library.
1 mL of a ~1011pfu/mL cyclic 7-amino acid (CX7C) peptide phage library using NNK codons in T7 phage was injected into the tail vein of Spraque-Dawley rats 5 days (9 animals) or 14 days (10 animals) after unilateral ONC. One hour after injection the animal was perfused with PBS to remove unbound phage, then both eyes were dissected, dissociated, and incubated with BLT5403 E. coli for several hours to pre-amplify retina-homing phage. The phages were then purified and the resulting library was amplified by PCR for Ion Torrent Sequencing. Sequencing data were filtered, sorted, and translated using custom Python software, and subsequent motif analysis was done in MATLAB using custom and previously published software. Potential retina-homing peptide motifs were isolated using a hierarchical clustering algorithm and tree-based distance metrics, and subsequently verified by inspection and comparison to known peptide motifs.
We identified a pool of tripeptide motifs conserved among phage captured by the retina, enriched at a level several thousand-fold over random phage selection (p<0.05). The control retina, 5d ONC retina, and 14d ONC retina gave rise to an overlapping motif pool, but each also contained unique sets of motifs that are putative homing motifs for targets specifically expressed in that group. Motifs most highly enriched in 5d and 14d ONC eyes included KGR, SKS, RSK, and PRK, RSR, RRS, respectively, while motifs most highly enriched in uninjured eyes included RGS, RKS, and GRK.
In vivo phage display revealed motifs among small peptide sequences that may correspond to retina-homing sequences, and may allow identification of reactive endothelium after ONC. These motifs may provide a backbone on which functional molecules or imaging probes can be delivered to reactive endothelium.
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