Dermatomyositis autoantigen CHD4 forms immune stimulatory complexes with endogenous DNA.

Dermatomyositis (DM) is an idiopathic inflammatory myopathy belonging to the spectrum of autoimmune connective tissue diseases. DM patients present with antinuclear antibodies against Mi-2, also known as Chromodomain-helicase-DNA-binding protein 4 (CHD4). CHD4 is upregulated in DM skin biopsies and could potentially affect DM pathophysiology as it binds endogenous DNA with a high affinity (KD = 0.2 nM ± 0.076 nM) and forms CHD4-DNA complexes. The complexes are localized in the cytoplasm of UV-radiated and transfected HaCaTs and amplify the expression of interferon (IFN) regulated genes and the amount of functional CXCL10 protein stronger than DNA alone. The enhancement of the type I IFN pathway activation in HaCaTs through CHD4-DNA signalling suggests a possible mechanism for the sustainment of the pro-inflammatory vicious cycle in DM skin lesions.

RNA Aptamers Recognizing Murine CCL17 Inhibit T Cell Chemotaxis and Reduce Contact Hypersensitivity In Vivo.

The chemokine CCL17, mainly produced by dendritic cells (DCs) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers, which were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 toward CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevented ear swelling and T cell infiltration into the ears of sensitized mice after challenge with the contact sensitizer. The results of this proof-of-principle study establish aptamers as potent inhibitors of CCL17-mediated chemotaxis. Potentially, CCL17-specific aptamers may be used therapeutically in humans to treat or prevent allergic and inflammatory diseases.

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

The click-SELEX procedure enables the identification of nucleobase-modified aptamers in which chemical entities are introduced by a copper(i)-catalysed alkyne-azide 'click' reaction. Here we report on the impact of modified nucleobases on PCR conditions and the average amount of modified nucleobases on click-SELEX performance. We demonstrate click-SELEX being strongly dependent on which and on how many modifications are used. However, when using C3-GFP the number of modifications did not impact the overall success of the selection procedure.

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

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.

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

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

Dynamic changes in DNA populations revealed by split-combine selection.

Clickmers are chemically modified aptamers representing an innovative reagent class for developing binders for biomolecules with great impact on therapeutic and diagnostic applications. To establish a novel layer for screening various chemical entities, we developed a split-combine selection strategy simultaneously enriching for clickmers having different modifications. Due to the inherent design of this strategy, dynamic changes of DNA populations are traceable at an individual sequence level. Besides off-rate guided enrichment, the process makes the survival of the sequences most adapted to the applied selection condition observable. The underlying strategy provides unprecedented molecular insight into the selection process, based on which more sophisticated procedures will become pliable in the future.

Customised nucleic acid libraries for enhanced aptamer selection and performance.

Aptamers are short single-stranded oligo(deoxy)nucleotides that are selected to bind to target molecules with high affinity and specificity. Because of their sophisticated characteristics and versatile applicability, aptamers are thought to become universal molecular probes in biotechnological and therapeutic applications. However, the variety of possible interactions with a putative target molecule is limited by the chemical repertoire of the natural nucleobases. Consequently, many desired targets are not addressable by aptamers. This obstacle is overcome by broadening the chemical diversity of aptamers, mainly achieved by nucleobase-modifications and the introduction of novel bases or base pairs. We discuss these achievements and the characteristics of the respective modified aptamers, reflected by SOMAmers (slow off-rate modified aptamers), clickmers, and aptamers bearing an expanded genetic alphabet.