Enrichment of Cell‐Targeting and Population‐Specific Aptamers by Fluorescence‐Activated Cell Sorting

Raddatz, Marie-Sophie L.; Dolf, Andreas; Endl, Elmar; Knolle, Percy; Famulok, Michael; Mayer, Günter

doi:10.1002/ange.200800216

Cited by 31 publications

(18 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Instead of using the tedious, time-consuming and radioactive EMSA process to monitor the progress of selection, FACS has been proven a simple, yet effective method of characterizing DNA-target binding affinity. [29][30][31][32][33][34] More importantly, during selection, by incubating Shp2-GST beads with the DNA library in binding buffer, proteins and DNA can interact in the solution phase; this allows effective binding to take place. Furthermore, beads can be washed thoroughly to remove nonspecific binders.…”

Section: Introductionmentioning

confidence: 99%

A G‐Quadruplex Aptamer Inhibits the Phosphatase Activity of Oncogenic Protein Shp2 in vitro

Liu

et al. 2011

ChemBioChem

View full text Add to dashboard Cite

Shp2 is a member of the protein tyrosine phosphatase (PTP) family, which regulates a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Using a recombinant Shp2-GST protein as the target and GST as a counter target, we have identified two classes of single-stranded DNA aptamers that selectively bind to Shp2 with a K(d) in the nanomolar range. Structural studies of the most abundant sequence in the enriched library, HJ24, revealed a parallel G-quadruplex as the core binding domain. Furthermore, this aptamer was found to be an effective inhibitor of Shp2 phosphatase, an effect which was readily reversed by using the cDNA of HJ24. In view of these characteristics, this aptamer has the potential to be used for further development of Shp2 assays and therapeutics for the treatment of Shp2-dependent cancers and other diseases.

show abstract

Section: Introductionmentioning

confidence: 99%

A G‐Quadruplex Aptamer Inhibits the Phosphatase Activity of Oncogenic Protein Shp2 in vitro

Liu

et al. 2011

ChemBioChem

View full text Add to dashboard Cite

show abstract

“…As probes for molecular recognition, the efficacy of DNA aptamers has been successfully demonstrated in many applications. [10,11] Aptamers are single-strand oligonucleotides derived from a selection process called systematic evolution of ligands by exponential enrichment (SELEX). In our study, the aptamers were selected from whole intact biological live cells through a process called cell-SELEX, [11] and these aptamers are capable of binding to their target molecules on the cell-membrane surface with high affinity and specificity.…”

Section: Introductionmentioning

confidence: 99%

“…[10,11] Aptamers are single-strand oligonucleotides derived from a selection process called systematic evolution of ligands by exponential enrichment (SELEX). In our study, the aptamers were selected from whole intact biological live cells through a process called cell-SELEX, [11] and these aptamers are capable of binding to their target molecules on the cell-membrane surface with high affinity and specificity. [12] In comparison with the molecular probes currently available for receptor recognition, such as monoclonal antibodies, aptamers offer significant advantages over existing antibody-based recognition procedures in that they offer higher binding affinity (higher retention/reduced dissociation) and specificity to the target (ability to determine variations on the protein target down to single amino acid changes), higher selectivity against mutated protein epitopes, and potentially reduced immunogenicity and increased tumor penetration associated with their size.…”

Section: Introductionmentioning

confidence: 99%

Mapping Receptor Density on Live Cells by Using Fluorescence Correlation Spectroscopy

Chen

Munteanu

Huang

et al. 2009

Chemistry A European J

View full text Add to dashboard Cite

Study of the density, spatial distribution, and molecular interactions of receptors on the cell membrane provides the knowledge required to understand cellular behavior and biological functions, as well as to discover, design, and screen novel therapeutic agents. However, the mapping of receptor distribution and the monitoring of ligand–receptor interactions on live cells in a spatially and temporally ordered manner are challenging tasks. In this paper, we apply fluorescence correlation spectroscopy (FCS) to map receptor densities on live cell membranes by introducing fluorescently marked aptamer molecules, which specifically bind to certain cell-surface receptors. The femtoliter-sized (0.4 fL) observation volume created by FCS allows fluorescent-aptamer detection down to 2 molecules and appears to be an ideal and highly sensitive biophysical tool for studying molecular interactions on live cells. Fluorophore-labeled aptamers were chosen for receptor recognition because of their high binding affinity and specificity. Aptamer sgc8, generated for specific cell recognition by a process called cell systematic evolution of ligands by exponential enrichment, was determined by FCS to have a binding affinity in the picomolar range (dissociation constant Kd = 790 ± 150 pM) with its target membrane receptor, human protein tyrosine kinase-7 (PTK7), a potential cancer biomarker. We then constructed a cellular model and applied this aptamer–receptor interaction to estimate receptor densities and distributions on the cell surface. Specifically, different expression levels of PTK7 were studied by using human leukemia CCRF-CEM cells (1300±190 receptors μm−2) and HeLa cervical cancer cells (550±90 receptors μm−2). Competition studies with excess nonlabeled aptamers and proteinase treatment studies proved the validity of the density-estimation approach. With its intrinsic advantages of direct measurement, high sensitivity, fast analysis, and single-cell measurement, this FCS density-estimation approach holds potential for future applications in molecular-interaction studies and density estimations for subcellular structures and membrane receptors.

show abstract

“…Diese sind in jeder Zellprä-paration vorhanden und können mit den klassischen Methoden (Waschen adhärenter Zellen oder Zentrifugation von Suspensionszellen) nicht entfernt werden. Da tote Zellen eine starke, Sequenz-unabhängige Affinität zu Nukleinsäuren aufweisen, führt dies mindestens zu einem ineffizienten Selektionsprozess [8] oder sogar zum Scheitern des Experiments [9], sodass sich Zell-SELEX bislang nicht als gängige Methode etablieren konnte.…”

Section: Zell-selexunclassified

“…Die Gruppen um Mayer und Famulok nutzten die Durchflusszytometrie nicht nur, um bindende von nicht bindenden Nukleinsäuren zu trennen, sondern auch um tote Zellen auszusortieren. Auf diese Weise erhielten sie B-Zellspezifische Aptamere, die sogar zwischen Zellpopulationen unterscheiden konnten [8].…”

Section: Zell-selexunclassified

Zellspezifische Aptamere in Diagnostik und Therapie

2011

View full text Add to dashboard Cite

Mittels Zell-SELEX lassen sich Aptamere selektieren, die intakte Zellen spezifisch erkennen. Anwendungen liegen in der Identifizierung von Zellpopulationen sowie in der Therapie, da zellspezifische Aptamere regulatorische Effekte auf Zielzellen ausüben oder Wirkstoffe vermitteln können.Cell-SELEX allows the selection of aptamers that specifically recognize live target cells. Applications include isolation and identification of cell populations or their constituents. Furthermore, some of these aptamers show regulatory effects or deliver specific drugs and may therefore be promising therapeutics.ó Aptamere sind kurze Nukleinsäuren (typischerweise zwölf bis 80 Nukleotide), die aufgrund ihrer räumlichen Struktur mit hoher Affinität und Spezifität diverse Zielmoleküle binden. Aptamere werden in vitro in einem SELEX-Prozess (systematic evolution of ligands by exponential enrichment) selektiert [1, 2]. Bei dieser Methode nutzt man eine Nukleinsäurebibliothek von enormer Diversität (bis zu 10 15 verschiedene DNA-oder RNA-Moleküle), von der man annimmt, dass diese einige Moleküle beinhaltet, die an das gewünsch-te Target binden. Die Targets, für die bereits erfolgreich Aptamere selektiert wurden, reichen von kleinen Molekülen über Peptide, Proteine und Viren bis hin zu ganzen Zellen [3].

show abstract

Enrichment of Cell‐Targeting and Population‐Specific Aptamers by Fluorescence‐Activated Cell Sorting

Cited by 31 publications

References 21 publications

A G‐Quadruplex Aptamer Inhibits the Phosphatase Activity of Oncogenic Protein Shp2 in vitro

A G‐Quadruplex Aptamer Inhibits the Phosphatase Activity of Oncogenic Protein Shp2 in vitro

Mapping Receptor Density on Live Cells by Using Fluorescence Correlation Spectroscopy

Zellspezifische Aptamere in Diagnostik und Therapie

Contact Info

Product

Resources

About