Cell-Internalization SELEX: Method for Identifying Cell-Internalizing RNA Aptamers for Delivering siRNAs to Target Cells

Abstract

After a decade of work to address cellular uptake, the principal obstacle to RNAi-based therapeutics, there is now well-deserved, renewed optimism about RNAi-based drugs. Phase I and II studies have shown safe, strong, and durable-gene knockdown (80–90 %, lasting for a month after a single injection) and/or clinical benefit in treating several liver pathologies. Although promising, these studies have also highlighted the need for robust delivery techniques to develop RNAi therapeutics for treating other organ systems and diseases. Conjugation of siRNAs to cell-specific, synthetic RNA ligands (aptamers) is being proposed as a viable solution to this problem. While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types. We describe a cell-based selection process for the rapid identification and characterization of RNA aptamers suited for delivering siRNA drugs into the cytoplasm of target cells. This process, termed “cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment),” entails the combination of multiple sophisticated technologies, including cell culture-based SELEX procedures, next-generation sequencing (NGS), and novel bioinformatics tools.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

“…The principal obstacle to RNAi-based therapeutics is cellular uptake. Cell-internalizing RNA aptamers conjugated with siRNAs as a delivery tool is being proposed to address this problem [40]. In 2012, Giangrande’s lab successively obtained aptamers that internalize into cells that express specific cell surface receptors (e.g., HER2 or TrkB) [41].…”

Ligand-Guided Selection of Target-Specific Aptamers: A Screening Technology for Identifying Specific Aptamers Against Cell-Surface Proteins

“…The bound DNA library was eluted by heating at 95°C for 10 min in 200 mL of DNAse/RNAse free water. A two-step PCR was employed for the optimization of the PCR conditions, and a large scale PCR was employed to expand the evolved library as reported elsewhere [12]. A doublestranded, PCR-amplified DNA library was made single stranded by using avidin agarose beads (Pierce) and desalted by using NAP-10 columns (GE) as described by Sefah et al [11].…”

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

“…However, such programs are not designed to manage high throughput data. To solve this drawback, Thiel et al use a series of software to analyze height rounds of a SELEX that were performed to identify aptamers able to internalize inside vascular smooth muscle (VSM) cells [29]. After removing all sequences that did not have more than three copies and present at least in two rounds, the frequency of 2312 unique sequences was measured at each round.…”