Method for Confirming Cytoplasmic Delivery of RNA Aptamers

Dickey, David D.; Thomas, Gregory S.; Dassie, Justin P.; Giangrande, Paloma H.

doi:10.1007/978-1-4939-3112-5_17

Cited by 7 publications

(8 citation statements)

References 31 publications

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“…Mix the reaction by gently pipetting up and down after 7.5 min has past. Start the timer after the NAAG solution is added to the first sample.Add 200 µL of ice cold 0.1 M Na 3 HPO 4 to each sample to stop the reaction.Prepare columns by placing as many Pasteur pipettes as needed into the plastic rack, with one 3 mm borosilicate glass ball in each pipette [15]. …”

Section: Methodsmentioning

confidence: 99%

“…Prepare columns by placing as many Pasteur pipettes as needed into the plastic rack, with one 3 mm borosilicate glass ball in each pipette [15]. …”

Section: Methodsmentioning

confidence: 99%

“…This will be of interest to those who wish to truncate other aptamers to PSMA or aptamers to other protein targets with carboxypeptidase activity. A detailed description of the NAALADase assay can be also found in Dickey et al 2016 [15]. …”

Section: Introductionmentioning

confidence: 99%

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Structural computational modeling of RNA aptamers

Dickey

Chen

et al. 2016

Methods

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RNA aptamers represent an emerging class of biologics that can be easily adapted for personalized and precision medicine. Several therapeutic aptamers with desirable binding and functional properties have been developed and evaluated in preclinical studies over the past 25 years. However, for the majority of these aptamers, their clinical potential has yet to be realized. A significant hurdle to the clinical adoption of this novel class of biologicals is the limited information on their secondary and tertiary structure. Knowledge of the RNA’s structure would greatly facilitate and expedite the post-selection optimization steps required for translation, including truncation (to reduce costs of manufacturing), chemical modification (to enhance stability and improve safety) and chemical conjugation (to improve drug properties for combinatorial therapy). Here we describe a structural computational modeling methodology that when coupled to a standard functional assay, can be used to determine key sequence and structural motifs of an RNA aptamer. We applied this methodology to enable the truncation of an aptamer to prostate specific membrane antigen (PSMA) with great potential for targeted therapy that had failed previous truncation attempts. This methodology can be easily applied to optimize other aptamers with therapeutic potential.

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Section: Methodsmentioning

confidence: 99%

“…Prepare columns by placing as many Pasteur pipettes as needed into the plastic rack, with one 3 mm borosilicate glass ball in each pipette [15]. …”

Section: Methodsmentioning

confidence: 99%

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Structural computational modeling of RNA aptamers

Dickey

Chen

et al. 2016

Methods

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“…Nuclease degradation is common to all DNA‐based structures. In order to circumvent that, chemical modifications such as fluoro‐, amino‐, O‐methyl bases were commonly applied . Some DNA‐based structures can resist nuclease action, but the circulation time of the objects needs to be extended due to renal filtration.…”

Section: Conclusion and Perspectivementioning

confidence: 99%

Programmable and Multifunctional DNA‐Based Materials for Biomedical Applications

et al. 2018

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DNA encodes the genetic information; recently, it has also become a key player in material science. Given the specific Watson-Crick base-pairing interactions between only four types of nucleotides, well-designed DNA self-assembly can be programmable and predictable. Stem-loops, sticky ends, Holliday junctions, DNA tiles, and lattices are typical motifs for forming DNA-based structures. The oligonucleotides experience thermal annealing in a near-neutral buffer containing a divalent cation (usually Mg ) to produce a variety of DNA nanostructures. These structures not only show beautiful landscape, but can also be endowed with multifaceted functionalities. This Review begins with the fundamental characterization and evolutionary trajectory of DNA-based artificial structures, but concentrates on their biomedical applications. The coverage spans from controlled drug delivery to high therapeutic profile and accurate diagnosis. A variety of DNA-based materials, including aptamers, hydrogels, origamis, and tetrahedrons, are widely utilized in different biomedical fields. In addition, to achieve better performance and functionality, material hybridization is widely witnessed, and DNA nanostructure modification is also discussed. Although there are impressive advances and high expectations, the development of DNA-based structures/technologies is still hindered by several commonly recognized challenges, such as nuclease instability, lack of pharmacokinetics data, and relatively high synthesis cost.

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“…By contrast, the methods available to test binding and internalization of aptamers identified through Cell-SELEX are much slower with less throughput. Currently used methods include quantitative PCR (qPCR), flow cytometry and fluorescent or confocal microscopy [8,25,26]. While qPCR has exquisite sensitivity, this method requires a large amount of starting material and extensive and time consuming sample processing with multiple steps, which leads to high experiment-to-experiment variability.…”

Section: Introductionmentioning

confidence: 99%

AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

Thiel

Giangrande

2016

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The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization.

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Method for Confirming Cytoplasmic Delivery of RNA Aptamers

Cited by 7 publications

References 31 publications

Structural computational modeling of RNA aptamers

Structural computational modeling of RNA aptamers

Programmable and Multifunctional DNA‐Based Materials for Biomedical Applications

AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

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