Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media

Hernandez, Frank J.; Stockdale, Katie R.; Huang, Lingyan; Horswill, Alexander R.; Behlke, Mark A.; McNamara, James O

doi:10.1089/nat.2011.0316

Cited by 36 publications

(33 citation statements)

References 43 publications

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“…Prior to starting a selection, cells should be tested for the desired phenotype/genotype (e.g., HER2 positivity) [20]. In addition, cells should be screened for mycoplasma contamination as this bacterium has been shown to secrete nucleases capable of degrading 2′-fluro-modified RNA aptamers [31].

Prepare nontarget and target cells : On day 1, seed both target and nontarget cells on 15 cm plates.

…”

Section: Methodsmentioning

confidence: 99%

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

Thiel

Flenker

et al. 2014

Methods in Molecular Biology

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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.

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Prepare nontarget and target cells : On day 1, seed both target and nontarget cells on 15 cm plates.

…”

Section: Methodsmentioning

confidence: 99%

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

Thiel

Flenker

et al. 2014

Methods in Molecular Biology

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“…Special DNA and RN A polymerases that are able to utilise nucleoside triphosphate substrates with a modified, for example, 2’ sugar position are used to generate such oligonucleotides. 2’-Amino pyrimidine nucleosides [20, 21], 2’-fluoropyrimidine nucleosides [22, 23], 2’-O-methyl purine, and 2’-O-methyl pyrimidine nucleosides [24, 25] are currently used for this purpose. The only aptamer approved for medical application known as Macugen (Fig.…”

Section: Limitations In Aptamer Application and Possible Sol Utionsmentioning

confidence: 99%

Aptamers: Problems, Solutions and Prospects

2013

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Aptamers are short single-stranded oligonucleotides that are capable of binding various molecules with high affinity and specificity. When the technology of aptamer selection was developed almost a quarter of a century ago, a suggestion was immediately put forward that it might be a revolutionary start into solving many problems associated with diagnostics and the therapy of diseases. However, multiple attempts to use aptamers in practice, although sometimes successful, have been generally much less efficient than had been expected initially. This review is mostly devoted not to the successful use of aptamers but to the problems impeding the widespread application of aptamers in diagnostics and therapy, as well as to approaches that could considerably expand the range of aptamer application.

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“…1) is time consuming, costly, and often fails to identify high-affinity aptamer due to PCR bias (PCR is used after each round to amplify the target bound aptamers). In several recent studies, highthroughput sequencing enabled the identification of high-affinity aptamers after many fewer rounds of selection, reducing time, cost, and PCR bias (12)(13)(14)(15). In the standard protocol, aptamers are selected against recombinant protein products in solution.…”

Section: Oligonucleotide Aptamer Ligandsmentioning

confidence: 99%

Use of Oligonucleotide Aptamer Ligands to Modulate the Function of Immune Receptors

Gilboa

McNamara

Pastor

2013

Clinical Cancer Research

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The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. The recent U.S. Food and Drug Administration approval of ipilumimab, an antibody that blocks the inhibitory action of CTLA-4, and clinical trials targeting 4-1BB and PD-1 or PD-L1, have underscored the therapeutic potential of using immunomodulatory antibodies to stimulate protective immunity in human patients. Nonetheless, systemic administration of immunomodulatory antibodies has been associated with dose-limiting autoimmune pathologies, conceivably reflecting also the activation of resident autoreactive T cells. Arguably, targeting immunomodulatory ligands to the disseminated tumor lesions of the patient would reduce such drug-associated toxicities. We have recently developed a new class of inhibitory (CTLA-4) and agonistic (4-1BB and OX-40) ligands composed of short oligonucleotide (ODN) aptamers that exhibited bioactivities comparable or superior to that of antibodies. To reduce toxicity, the immunomodulatory aptamers were targeted to the tumor by conjugation to a second aptamer that bound to a product expressed on the surface of the tumor cell, the targeting aptamer, generating a bispecific aptamer conjugate analogous to bispecific antibodies. In a proof-of-concept study in mice, we have shown that an agonistic 4-1BB-binding aptamer conjugated to a prostate-specific membrane antigen (PSMA)-binding aptamer led to the inhibition of PSMA-expressing tumors, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared with nontargeted costimulation with 4-1BB antibodies or 4-1BB aptamers. The cell-free chemically synthesized ODN aptamers offer significant advantages over antibodies in terms of synthesis, cost, as well as conjugation chemistry needed to generate bispecific ligand fusions.

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Degradation of Nuclease-Stabilized RNA Oligonucleotides in Mycoplasma-Contaminated Cell Culture Media

Cited by 36 publications

References 43 publications

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

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

Aptamers: Problems, Solutions and Prospects

Use of Oligonucleotide Aptamer Ligands to Modulate the Function of Immune Receptors

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