DNA Subtraction of In Vivo Selected Phage Repertoires for Efficient Peptide Pathology Biomarker Identification in Neuroinflammation Multiple Sclerosis Model

Abstract

To streamline in vivo biomarker discovery, we developed a suppression subtractive DNA hybridization technique adapted for phage-displayed combinatorial libraries of 12 amino acid peptides (PhiSSH). Physical DNA subtraction is performed in a one-tube-all-reactions format by sequential addition of reagents, producing the enrichment of specific clones of one repertoire. High-complexity phage repertoires produced by in vivo selections in the multiple sclerosis rat model (experimental autoimmune encephalomyelitis, EAE) and matched healthy control rats were used to evaluate the technique. The healthy repertoire served as a physical DNA subtractor from the EAE repertoire to produce the subtraction repertoire. Full next-generation sequencing (NGS) of the three repertoires was performed to evaluate the efficiency of the subtraction technique. More than 96% of the clones common to the EAE and healthy repertoires were absent from the subtraction repertoire, increasing the probability of randomly selecting various specific peptides for EAE pathology to about 70%. Histopathology experiments were performed to confirm the quality of the subtraction repertoire clones, producing distinct labeling of the blood–brain barrier (BBB) affected by inflammation among healthy nervous tissue or the preferential binding to IL1-challenged vs. resting human BBB model. Combining PhiSSH with NGS will be useful for controlled in vivo screening of small peptide combinatorial libraries to discover biomarkers of specific molecular alterations interspersed within healthy tissues.

Screening for Interacting Proteins with Peptide Biomarker of Blood–Brain Barrier Alteration under Inflammatory Conditions

“…The technology of the in vivo selection of phage-displayed peptides is a valuable tool for the identification of specific ligands in tissue sites under pathological conditions such as cancer and neuroinflammation [ 15 , 16 , 17 , 18 ]. In vivo selections mainly target the vascular system, presenting both disease-altered targets and unaffected molecules.…”

“…Although, other useful methodologies for target identification and validation exist and have been tested in neurodegenerative and inflammatory conditions [ 19 ], such as high-throughput screening, which is advantageous when little is known of a target, allowing the assay and screening of a large number of biological effectors and modulators against designated and exclusive targets [ 20 ]. The screening and elimination of common peptide ligands has resulted in the discovery of peptide binding inflammatory specific targets [ 17 ] using a phage-displayed library of 12 peptides to select those bound to blood vessels in the CNS parenchyma of rats with experimental autoimmune encephalomyelitis (EAE) [ 17 ]. EAE is a model of neuroinflammation that mimics significant neuropathological aspects of multiple sclerosis [ 17 , 21 ].…”

“…The screening and elimination of common peptide ligands has resulted in the discovery of peptide binding inflammatory specific targets [ 17 ] using a phage-displayed library of 12 peptides to select those bound to blood vessels in the CNS parenchyma of rats with experimental autoimmune encephalomyelitis (EAE) [ 17 ]. EAE is a model of neuroinflammation that mimics significant neuropathological aspects of multiple sclerosis [ 17 , 21 ]. BBB disruption is one of the EAE characteristics, occurring due to the abnormal expression of endothelial cell proteins, which results in perivascular infiltration of peripheral immune cells and the development of focal inflammatory lesions [ 22 , 23 ].…”

“…Among several phage-displayed peptides selected in the EAE animal model, we have identified specific peptides capable of labeling the human endothelial cell line HCMEC/D3 under proinflammatory conditions in in vivo binding studies. One of these peptides expressed by the phage clone 88 showed high specific affinity under inflammatory conditions with the endothelial cell line under IL-1β exposure [ 17 ]. The purpose of the present study is to validate the interaction of the peptide-88 with its possible targets expressed under inflammatory conditions by using nano-scale liquid chromatographic tandem MS (nLC-MS) of the cross-linked peptide to target proteins.…”

Phage display peptide libraries: deviations from randomness and correctives

“…Not only did the amino acids previously over represented, drop to a reasonable expected base line, but the correction for G overuse reduced the UAG concentration as well. To assess the corrections taken to improve the randomness of our libraries we analysed the profile produced for the commercially available New England BioLabs (NEB) Ph.D.-7 library (see, for example ( 2 , 29 , 47 , 49 , 53 , 57 , 63 , 65 , 66 , 72–75 )). For this, we have compared the nucleotide and amino-acid distributions of the NEB library with that observed in our fourth generation unbiased library (Figure 1D ).…”