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Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.

Hernandez LI, Flenker KS, Hernandez FJ, Klingelhutz AJ, McNamara JO, Giangrande PH - Pharmaceuticals (Basel) (2013)

Bottom Line: Here we describe methods to monitor for cellular uptake of aptamers.These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay.Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

No MeSH data available.


Schematic of in vitro-Systematic Evolution of Ligands by EXponential enrichment (SELEX; upper panel) and Cell-Internalization SELEX methodologies (lower panel). For in vitro-SELEX, a library of RNA molecules is incubated with the target (e.g. recombinant protein) (Step 1) and binders are separated from non-binders (Step 2). Recovered binders (Step 3) are subsequently amplified (Step 4) to generate a pool of molecules that displays high affinity and specificity for the target. For cell-internalization SELEX, a library of RNA molecules is incubated with non-target cells (counter selection) (Step 1) and those RNAs that do not bind (supernatant) are then transferred to the target cells (Step 2). The cell incubations are performed at 37 °C to facilitate cell internalization (Step 3). A stringent wash step (high salt wash, trypsin wash) is then performed to remove unbound and/or surface bound RNA. The internalized RNA is recovered using TRIzol extraction (Step 4) and amplified by PCR (Step 5). The process is repeated several more times to enrich for RNA sequences with cell-specific, internalizing properties (Step 6). The enriched pool of RNA is then subjected to cloning and sequencing (Step 7).
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pharmaceuticals-06-00295-f001: Schematic of in vitro-Systematic Evolution of Ligands by EXponential enrichment (SELEX; upper panel) and Cell-Internalization SELEX methodologies (lower panel). For in vitro-SELEX, a library of RNA molecules is incubated with the target (e.g. recombinant protein) (Step 1) and binders are separated from non-binders (Step 2). Recovered binders (Step 3) are subsequently amplified (Step 4) to generate a pool of molecules that displays high affinity and specificity for the target. For cell-internalization SELEX, a library of RNA molecules is incubated with non-target cells (counter selection) (Step 1) and those RNAs that do not bind (supernatant) are then transferred to the target cells (Step 2). The cell incubations are performed at 37 °C to facilitate cell internalization (Step 3). A stringent wash step (high salt wash, trypsin wash) is then performed to remove unbound and/or surface bound RNA. The internalized RNA is recovered using TRIzol extraction (Step 4) and amplified by PCR (Step 5). The process is repeated several more times to enrich for RNA sequences with cell-specific, internalizing properties (Step 6). The enriched pool of RNA is then subjected to cloning and sequencing (Step 7).

Mentions: Isolation of aptamers with affinity and specificity for a target of interest involves iterative rounds of affinity purification and amplification via a process termed in vitro-Systematic Evolution of Ligands by EXponential enrichment (SELEX) [14,15] (Figure 1; top panel). While in vitro-SELEX results in aptamers with affinities and specificities for their cognate targets comparable to those seen with antibodies, this process does not guarantee the identification of aptamers capable of recognizing their target in the context of the cell membrane and of internalizing into the target cell. We have recently described a novel cell-based selection strategy that we refer to as cell-internalization SELEX for isolating aptamers that internalize upon binding to their cognate receptor [16,17,18] (Figure 1; bottom panel). Cell-internalization SELEX has several advantages over other selection approaches for targeted therapeutic applications: (1) it favors the isolation of RNAs that bind to receptors in their native state; and (2) it enriches for RNAs capable of entering the target cell. To date, this approach has yielded aptamers capable of internalizing into HER2-positive mammary carcinoma cells [16], vascular smooth muscle cells [17] and TrkB-expressing cells [18]. Importantly, we demonstrated that when conjugated to therapeutic siRNAs, the cell-internalizing aptamers were capable of delivering their cargo to the cytoplasm of the target cells resulting in a robust RNAi response [16].


Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.

Hernandez LI, Flenker KS, Hernandez FJ, Klingelhutz AJ, McNamara JO, Giangrande PH - Pharmaceuticals (Basel) (2013)

Schematic of in vitro-Systematic Evolution of Ligands by EXponential enrichment (SELEX; upper panel) and Cell-Internalization SELEX methodologies (lower panel). For in vitro-SELEX, a library of RNA molecules is incubated with the target (e.g. recombinant protein) (Step 1) and binders are separated from non-binders (Step 2). Recovered binders (Step 3) are subsequently amplified (Step 4) to generate a pool of molecules that displays high affinity and specificity for the target. For cell-internalization SELEX, a library of RNA molecules is incubated with non-target cells (counter selection) (Step 1) and those RNAs that do not bind (supernatant) are then transferred to the target cells (Step 2). The cell incubations are performed at 37 °C to facilitate cell internalization (Step 3). A stringent wash step (high salt wash, trypsin wash) is then performed to remove unbound and/or surface bound RNA. The internalized RNA is recovered using TRIzol extraction (Step 4) and amplified by PCR (Step 5). The process is repeated several more times to enrich for RNA sequences with cell-specific, internalizing properties (Step 6). The enriched pool of RNA is then subjected to cloning and sequencing (Step 7).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3722562&req=5

pharmaceuticals-06-00295-f001: Schematic of in vitro-Systematic Evolution of Ligands by EXponential enrichment (SELEX; upper panel) and Cell-Internalization SELEX methodologies (lower panel). For in vitro-SELEX, a library of RNA molecules is incubated with the target (e.g. recombinant protein) (Step 1) and binders are separated from non-binders (Step 2). Recovered binders (Step 3) are subsequently amplified (Step 4) to generate a pool of molecules that displays high affinity and specificity for the target. For cell-internalization SELEX, a library of RNA molecules is incubated with non-target cells (counter selection) (Step 1) and those RNAs that do not bind (supernatant) are then transferred to the target cells (Step 2). The cell incubations are performed at 37 °C to facilitate cell internalization (Step 3). A stringent wash step (high salt wash, trypsin wash) is then performed to remove unbound and/or surface bound RNA. The internalized RNA is recovered using TRIzol extraction (Step 4) and amplified by PCR (Step 5). The process is repeated several more times to enrich for RNA sequences with cell-specific, internalizing properties (Step 6). The enriched pool of RNA is then subjected to cloning and sequencing (Step 7).
Mentions: Isolation of aptamers with affinity and specificity for a target of interest involves iterative rounds of affinity purification and amplification via a process termed in vitro-Systematic Evolution of Ligands by EXponential enrichment (SELEX) [14,15] (Figure 1; top panel). While in vitro-SELEX results in aptamers with affinities and specificities for their cognate targets comparable to those seen with antibodies, this process does not guarantee the identification of aptamers capable of recognizing their target in the context of the cell membrane and of internalizing into the target cell. We have recently described a novel cell-based selection strategy that we refer to as cell-internalization SELEX for isolating aptamers that internalize upon binding to their cognate receptor [16,17,18] (Figure 1; bottom panel). Cell-internalization SELEX has several advantages over other selection approaches for targeted therapeutic applications: (1) it favors the isolation of RNAs that bind to receptors in their native state; and (2) it enriches for RNAs capable of entering the target cell. To date, this approach has yielded aptamers capable of internalizing into HER2-positive mammary carcinoma cells [16], vascular smooth muscle cells [17] and TrkB-expressing cells [18]. Importantly, we demonstrated that when conjugated to therapeutic siRNAs, the cell-internalizing aptamers were capable of delivering their cargo to the cytoplasm of the target cells resulting in a robust RNAi response [16].

Bottom Line: Here we describe methods to monitor for cellular uptake of aptamers.These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay.Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

No MeSH data available.