Dataset Information

SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging.

ABSTRACT: The SRB-2 aptamer originally selected against sulforhodamine B is shown here to promiscuously bind to various dyes with different colors. Binding of SRB-2 to these dyes results in either fluorescence increase or decrease, making them attractive for fluorescence microscopy and biological assays. By systematically varying fluorophore structural elements and measuring dissociation constants, the principles of fluorophore recognition by SRB-2 were analyzed. The obtained structure-activity relationships allowed us to rationally design a novel, bright, orange fluorescent turn-on probe (TMR-DN) with low background fluorescence, enabling no-wash live-cell RNA imaging. This new probe improved the signal-to-background ratio of fluorescence images by one order of magnitude over best previously known probe for this aptamer. The utility of TMR-DN is demonstrated by imaging ribosomal and messenger RNAs, allowing the observation of distinct localization patterns in bacteria and mammalian cells. The SRB-2 / TMR-DN system is found to be orthogonal to the Spinach/DFHBI and MG/Malachite green aptamer/dye systems.

SUBMITTER: Sunbul M

PROVIDER: S-EPMC6182184 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging.

Sunbul Murat M Jäschke Andres A

Nucleic acids research 20181001 18

The SRB-2 aptamer originally selected against sulforhodamine B is shown here to promiscuously bind to various dyes with different colors. Binding of SRB-2 to these dyes results in either fluorescence increase or decrease, making them attractive for fluorescence microscopy and biological assays. By systematically varying fluorophore structural elements and measuring dissociation constants, the principles of fluorophore recognition by SRB-2 were analyzed. The obtained structure-activity relationsh ...[more]

PMID: 29931157

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Project description:OBJECTIVE:Epidermal growth factor receptor (EGFR) is a theranostic biomarker for a variety of cancer types. The aim of the present study was to develop an 18F radiolabeled EGFR targeting RNA aptamer, and to investigate its ability to visualize and quantify EGFR in xenograft models. METHODS:Biolayer interferometry binding assay was used to detect the binding affinity of the alkyne-modified EGFR aptamer MinE07 (denoted as ME07) with recombinant human wild-type EGFR protein and the mutant EGFRvIII protein. Cy5-conjugated ME07 was used for flow cytometry and immunofluorescence staining, and an Alexa Fluor 488-labeled EGFR antibody (ab193244) was used as a control. 18F-Fluorobenzoyl (FB) azide was employed as a synthon to produce 18F-FB-ME07 via click chemistry, and the cellular uptake and internalization characteristics of 18F-FB-ME07 were investigated. Static PET scans, 60-min dynamic scans, and biodistribution study of 18F-FB-ME07 were performed in three types of tumor models. RESULTS:The Kd values of ME07 to wtEGFR and EGFRvIII proteins were 0.3 nM and 271 nM respectively. The A431, U87MG, and HCT-116 cells showed strong, weak, and negative binding with Cy5-ME07, which is consistent with EGFR expression level in these cells. Peak cell uptake values of 18F-FB-ME07 in A431, U87MG and HCT-116 cells were 2.86%, 2.19% and 0.88% of the added dose respectively. The mean internalization of 18F-FB-ME07 in these cells were 60.02%, 53.1%, and 52.8% of the total accumulated radioactivity. In static PET imaging, despite high uptake in the liver and kidneys, 18F-FB-ME07 showed reasonable accumulation in A431 tumors (1.02?±?0.13 %ID/g at 30 min after injection). Of note, the uptake of 18F-FB-ME07 in A431 xenografts was significantly higher than that in U87MG and HCT-116 xenografts. In A431 xenografted mice, the tumor/blood ratio was 3.89 and the tumor/muscle ratio reached 8.65. CONCLUSIONS:We for the first time generated an aptamer-derived EGFR targeting PET tracer 18F-FB-ME07, which showed highly selective targeting ability in mouse tumor models expressing different levels of EGFR. Our results suggest that 18F-FB-ME07 is a potential EGFR targeting molecular imaging probe for future clinical translation.

Dataset Information

SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging.

Publications

SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging.

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