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Facile supermolecular aptamer inhibitors of L-selectin.

Chang EK, Eckert MA, Ali MM, Riazifar H, Pone EJ, Liu L, Zhao W - PLoS ONE (2015)

Bottom Line: We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands.Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible.We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Sue and Bill Gross Stem Cell Research Center, Chao Family Comprehensive Cancer Center, Edwards Lifesciences Center for Advanced Cardiovascular Technology, Edwards Lifesciences Center for Advanced Cardiovascular Technology, Department of Biomedical Engineering, and Department of Pharmaceutical Sciences, University of California Irvine, Irvine, California, 92697, United States of America.

ABSTRACT
Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

No MeSH data available.


Related in: MedlinePlus

LS-Multi-Aptamer blocks binding to endogenous ligands without inducing shedding.(A) Relative percentage of bound SLeX following incubation of Jurkat cells with the indicated monovalent aptamers and Multi-Aptamers. (B) ELISA quantification of shed L-selectin following treatment of Jurkat cells with the indicated Multi-Aptamers or PMA. (C) Western blot analysis of Jurkat cell-associated L-selectin following the indicated treatments. Error bars are SEM.
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pone.0123034.g006: LS-Multi-Aptamer blocks binding to endogenous ligands without inducing shedding.(A) Relative percentage of bound SLeX following incubation of Jurkat cells with the indicated monovalent aptamers and Multi-Aptamers. (B) ELISA quantification of shed L-selectin following treatment of Jurkat cells with the indicated Multi-Aptamers or PMA. (C) Western blot analysis of Jurkat cell-associated L-selectin following the indicated treatments. Error bars are SEM.

Mentions: Due to the strong affinities of the Multi-Aptamer for L-selectin, we next explored its potential to modulate L-selectin function by inhibiting binding to endogenous ligands. One of the most well-established ligands for L-selectin is SLeX, which directly interacts with L-selectin and plays key roles in mediating initial tethering and rolling of leukocytes [36]. To test this hypothesis, we incubated 200,000 Jurkat cells with 100 nM of SC- or LS-Multi-Aptamer or the SC- or LS-Aptamer for 30 minutes prior to incubation with 50 μg/ml of FITC-labeled SLeX for 1 hour. Fluorescence was assessed via flow cytometry and normalized to untreated cells. Despite some off-target inhibition of Jurkat cell-SLeX binding in the presence of the SC-Multi-Aptamer, the LS-Multi-Aptamer still led to a dramatic reduction in Jurkat cell interaction with SLeX that was much more robust than inhibition with monovalent aptamers (Fig 6A). This data suggests that the LS-Multi-Aptamer can inhibit binding to endogenous ligands, either through blocking or inducing shedding of L-selectin from the cell surface.


Facile supermolecular aptamer inhibitors of L-selectin.

Chang EK, Eckert MA, Ali MM, Riazifar H, Pone EJ, Liu L, Zhao W - PLoS ONE (2015)

LS-Multi-Aptamer blocks binding to endogenous ligands without inducing shedding.(A) Relative percentage of bound SLeX following incubation of Jurkat cells with the indicated monovalent aptamers and Multi-Aptamers. (B) ELISA quantification of shed L-selectin following treatment of Jurkat cells with the indicated Multi-Aptamers or PMA. (C) Western blot analysis of Jurkat cell-associated L-selectin following the indicated treatments. Error bars are SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4380364&req=5

pone.0123034.g006: LS-Multi-Aptamer blocks binding to endogenous ligands without inducing shedding.(A) Relative percentage of bound SLeX following incubation of Jurkat cells with the indicated monovalent aptamers and Multi-Aptamers. (B) ELISA quantification of shed L-selectin following treatment of Jurkat cells with the indicated Multi-Aptamers or PMA. (C) Western blot analysis of Jurkat cell-associated L-selectin following the indicated treatments. Error bars are SEM.
Mentions: Due to the strong affinities of the Multi-Aptamer for L-selectin, we next explored its potential to modulate L-selectin function by inhibiting binding to endogenous ligands. One of the most well-established ligands for L-selectin is SLeX, which directly interacts with L-selectin and plays key roles in mediating initial tethering and rolling of leukocytes [36]. To test this hypothesis, we incubated 200,000 Jurkat cells with 100 nM of SC- or LS-Multi-Aptamer or the SC- or LS-Aptamer for 30 minutes prior to incubation with 50 μg/ml of FITC-labeled SLeX for 1 hour. Fluorescence was assessed via flow cytometry and normalized to untreated cells. Despite some off-target inhibition of Jurkat cell-SLeX binding in the presence of the SC-Multi-Aptamer, the LS-Multi-Aptamer still led to a dramatic reduction in Jurkat cell interaction with SLeX that was much more robust than inhibition with monovalent aptamers (Fig 6A). This data suggests that the LS-Multi-Aptamer can inhibit binding to endogenous ligands, either through blocking or inducing shedding of L-selectin from the cell surface.

Bottom Line: We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands.Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible.We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Sue and Bill Gross Stem Cell Research Center, Chao Family Comprehensive Cancer Center, Edwards Lifesciences Center for Advanced Cardiovascular Technology, Edwards Lifesciences Center for Advanced Cardiovascular Technology, Department of Biomedical Engineering, and Department of Pharmaceutical Sciences, University of California Irvine, Irvine, California, 92697, United States of America.

ABSTRACT
Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

No MeSH data available.


Related in: MedlinePlus