In Vitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease

Abstract

Chronic kidney disease (CKD) is a progressive pathological condition marked by a gradual loss of kidney function. Treatment of CKD is most effective when diagnosed at an early stage and patients are still asymptomatic. However, current diagnostic biomarkers (e.g., serum creatinine and urine albumin) are insufficient for prediction of the pathogenesis of the disease. To address this need, we applied a cell-SELEX (systematic evolution of ligands by exponential enrichment) approach and identified a series of DNA aptamers, which exhibit high affinity and selectivity for cytokine-stimulated cells, resembling some aspects of a CKD phenotype. The cell-SELEX approach was driven toward the enrichment of aptamers that internalize via the endosomal pathway by isolating the endosomal fractions in each selection cycle. Indeed, we demonstrated co-localization of selected aptamers with lysosomal-associated membrane protein 1 (LAMP-1), a late endosomal and lysosomal marker protein, by fluorescence in situ hybridization. These findings are consistent with binding and subsequent internalization of the aptamers into cytokine-stimulated cells. Thus, our study sets the stage for applying selected DNA aptamers as theragnostic reagents for the development of targeted therapies to combat CKD.

Aptamers: Uptake mechanisms and intracellular applications

“…With the fluorescence confocal microscopy, the subcellular distribution of internalized aptamers was characterized in four studies. The colocalization of cell type specific-DNA aptamers with LAMP-1, a late endosomal and lysosomal marker, was confirmed in renal proximal tubule RPTEC/TERT1 cells by in situ fluorescence hybridization [13]. Glioma-specific DNA aptamers bound Ku 70 and Ku 80 were found to colocalize in lysosomes, endoplasmic reticulum (ER), and Golgi apparatus with organelle-specific fluorescent dyes on live cell images by confocal microscopy [14].…”

Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines

“…This work shows that we can deliver functional RNA into cells and that it retains its folding and function once it gets there. Although endosomal escape is an essential step in many therapeutically relevant applications, such as RNAi-mediated gene silencing, other strategies aim at targeting endosomes directly 64 , 65 . The endosomal compartment is not a simple transient environment for the degradation or recycling of cell-surface receptors and uptake of nutrients.…”

Aptamer Cell-Based Selection: Overview and Advances

“…The use of an RNAse cocktail to remove surface-binding aptamers and recover cell-internalizing aptamers was more recently proposed [ 59 ]. Further, an interesting protocol to successfully enrich for aptamers that internalize via the endosomal pathway has been proposed by Ranches et al [ 60 ]. The proposed strategy is based on the isolation of the endosomal fractions by cell homogenization, followed by sucrose gradient centrifugation at each selection cycle.…”