Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection

Abstract

BackgroundThe broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.Methodology/Principal FindingsWe demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.Conclusions and SignificanceWe describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.

Manipulating the in vivo immune response by targeted gene knockdown

“…With high throughput sequencing and bioinformatics, enriched sequences can often be identified after only 5–6 rounds of selection with a much improved success rate [18–20]. Sequences that might have been lost because of bias in PCR amplification are retained.…”

Analyzing HT-SELEX data with the Galaxy Project tools – A web based bioinformatics platform for biomedical research

“…Originally, the precise sequences of aptamers from the final round of selection was obtained using sub-cloning techniques followed by sequencing each clone one at a time. High-throughput sequencing (HTS) fundamentally changed the aptamer field by enabling the sequencing of hundreds of millions of reads from multiple rounds of a selection [5-8]. With aptamer HTS data came the need for more sophisticated aptamer bioinformatics methods [9].…”

Cell-targeting aptamers act as intracellular delivery vehicles

“…With the basic developments in SELEX, different strategies have been demonstrated for generating cell-internalizing aptamers, such as whole-cell SELEX (also termed cellinternalization SELEX or cell-SELEX) and in vivo SELEX (Mi et al 2010;Thiel et al 2012;Zhou et al 2012;Cheng et al 2013;Gourronc et al 2013;Camorani et al 2014;Yan 2014). These SELEX processes target membrane/surfacebound antigens, leading to the production of internalizing aptamers.…”

“…Distinct surface Baptatopes^are involved in the SELEX process, and the selected aptamer will be internalized upon binding to the surface targets on the cell. It should be noted that with cell-internalization SELEX, there is a possibility of generating aptamers against multiple targets on the single cell (Thiel et al, 2012).…”

“…A specific aptamer targeting the ectodomain of a human plateletderived growth factor receptor has been generated to inhibit receptor signaling, thereby inhibiting glioblastoma cancer growth (Camorani et al 2014). Thiel et al (2012) have introduced bioinformatics-based cell-internalization SELEX, using vascular smooth muscle cell as the model.…”