Split–Combine Click-SELEX Reveals Ligands Recognizing the Transplant Rejection Biomarker CXCL9

Abstract

Renal rejection is a major incidence in patients after kidney transplantation and associated with allograft scarring and function loss, especially in antibody-mediated rejection. Regular clinical monitoring of kidney-transplanted patients is thus necessary, but measuring donor-specific antibodies is not always predictive, and graft biopsies are time-consuming and costly and may come up with a histological result unsuspicious for rejection. Therefore, a noninvasive diagnostic approach to estimate an increased probability of kidney graft rejection by measuring specific biomarkers is highly desired. The chemokine CXCL9 is described as an early indicator of rejection. In this work, we identified clickmers and an aptamer by split−combine click-SELEX (systematic evolution of ligands by exponential enrichment) that bind CXLC9 with high affinity. The aptamers recognize native CXCL9 and maintain binding properties under urine conditions. These features render the molecules as potential binding and detector probes for developing point-of-care devices, e.g., lateral flow assays, enabling the noninvasive monitoring of CXCL9 in renal allograft patients.

Dependence of click-SELEX performance on the nature and average number of modified nucleotides

“…Previously, we employed several azides for CuAAC to modify DNA libraries and subjected these to click-SELEX procedures. Among them, we used indole, benzyl, and other aromatic residues and successfully identified clickmers binding to cycle 3 Green Fluorescent Protein (C3-GFP), 5 streptavidin, 14 CXCL9, 15 and Δ 9 -tetrahydrocannabinol. 16 To more systematically investigate the suitability of individual azide moieties for the click-SELEX procedure, we performed click-SELEX targeting C3-GFP using eight different representative and available azides ( Fig.…”

“…However, currently, the methodology is limited to one modification per DNA strand but has been shown to be adaptable to multiplex formats, thereby assaying several chemical entities simultaneous. 14,15 Here we describe the suitability of different chemical entities for being used in the click-SELEX procedure, the impact of modifications on PCR performance, and the relation of selection output on the statistical number of modifications per DNA strand in the starting library. Our experiments reveal that the PCR performance is more reliable when using less modifications per DNA sequence.…”

An Antibody-Aptamer-Hybrid Lateral Flow Assay for Detection of CXCL9 in Antibody-Mediated Rejection after Kidney Transplantation

“…In parallel, a traditional solely antibody-based LFA yielded only a sensitivity of 53% with comparable specificity of 71% [ 70 ]. We suggest that the relatively smaller size of the 82 bp aptamer (size approximately 26.2 kDa [ 71 ]) could be a key point for the improved sensitivity especially with respect to the small target protein CXCL9 (11.7 kDa [ 72 ]). A second antibody as coupling partner on the nanoparticles as detection molecule could also impose a relevant steric hindrance due to its size (mean size of an IgG-antibody: ~150 kDs), possibly not offering the optimal binding pocket for the small CXCL9 molecule.…”

Controlled enzymatic synthesis of oligonucleotides

“…For instance, oligonucleotides are important building blocks in DNA-encoded libraries (DELs) for drug discovery campaigns 1,2 , storage of digital information 3,4 , and computing 5 . In addition, DNA and RNA can serve as potent catalysts [6][7][8][9][10] , binders [11][12][13][14][15][16][17] , and therapeutic agents 18,19 . The increasing popularity of nucleic acids is accompanied by an important surge in the demand of synthetic oligonucleotides, particularly of sequences containing chemical modifications.…”