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RNA aptamers that functionally interact with green fluorescent protein and its derivatives.

Shui B, Ozer A, Zipfel W, Sahu N, Singh A, Lis JT, Shi H, Kotlikoff MI - Nucleic Acids Res. (2011)

Bottom Line: These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness.Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH.FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways.

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Related in: MedlinePlus

Binding of FBPAs to GFPs and spectrally shifted GFP variants. (A) Comparison of G16 and AP3 binding to FPs show that AP3 binds GFP and EGFP with higher affinity than G16, and that binding to the coral protein Azami-green is of much lower affinity. (B) Filter-binding assay shows similar affinity of AP3 to GFP-related proteins. (C) Hill plot fits of data shown in (A) and (B) show similar affinity of AP3 to GFP-related proteins.
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gkr1264-F2: Binding of FBPAs to GFPs and spectrally shifted GFP variants. (A) Comparison of G16 and AP3 binding to FPs show that AP3 binds GFP and EGFP with higher affinity than G16, and that binding to the coral protein Azami-green is of much lower affinity. (B) Filter-binding assay shows similar affinity of AP3 to GFP-related proteins. (C) Hill plot fits of data shown in (A) and (B) show similar affinity of AP3 to GFP-related proteins.

Mentions: We next compared the binding of G16 and AP3 to GFP and other FPs. Aequorea victoria-derived fluorescent proteins are highly homologous at the level of primary sequence and overall structure, with EGFP differing only by the F64L and S65T amino acid substitutions (26), and yellow and cyan derivatives differing by five and four residues from EGFP, respectively (27,28). As shown in Figure 2A, both aptamers bound GFP and EGFP with roughly equivalent affinity, with AP3 demonstrating an ~5-fold greater affinity for both proteins relative to G16. Moreover, both aptamers bound the coral protein Azami-Green, which shares the overall 11-stranded β-barrel structure but diverges considerably in primary sequence (29), albeit with markedly lower affinity. Importantly, AP3 binds GFP and its derivatives with high affinity (Figure 2A–C); binding of AP3 to this broad range of commonly used FPs markedly increases its potential utility.Figure 2.


RNA aptamers that functionally interact with green fluorescent protein and its derivatives.

Shui B, Ozer A, Zipfel W, Sahu N, Singh A, Lis JT, Shi H, Kotlikoff MI - Nucleic Acids Res. (2011)

Binding of FBPAs to GFPs and spectrally shifted GFP variants. (A) Comparison of G16 and AP3 binding to FPs show that AP3 binds GFP and EGFP with higher affinity than G16, and that binding to the coral protein Azami-green is of much lower affinity. (B) Filter-binding assay shows similar affinity of AP3 to GFP-related proteins. (C) Hill plot fits of data shown in (A) and (B) show similar affinity of AP3 to GFP-related proteins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3300005&req=5

gkr1264-F2: Binding of FBPAs to GFPs and spectrally shifted GFP variants. (A) Comparison of G16 and AP3 binding to FPs show that AP3 binds GFP and EGFP with higher affinity than G16, and that binding to the coral protein Azami-green is of much lower affinity. (B) Filter-binding assay shows similar affinity of AP3 to GFP-related proteins. (C) Hill plot fits of data shown in (A) and (B) show similar affinity of AP3 to GFP-related proteins.
Mentions: We next compared the binding of G16 and AP3 to GFP and other FPs. Aequorea victoria-derived fluorescent proteins are highly homologous at the level of primary sequence and overall structure, with EGFP differing only by the F64L and S65T amino acid substitutions (26), and yellow and cyan derivatives differing by five and four residues from EGFP, respectively (27,28). As shown in Figure 2A, both aptamers bound GFP and EGFP with roughly equivalent affinity, with AP3 demonstrating an ~5-fold greater affinity for both proteins relative to G16. Moreover, both aptamers bound the coral protein Azami-Green, which shares the overall 11-stranded β-barrel structure but diverges considerably in primary sequence (29), albeit with markedly lower affinity. Importantly, AP3 binds GFP and its derivatives with high affinity (Figure 2A–C); binding of AP3 to this broad range of commonly used FPs markedly increases its potential utility.Figure 2.

Bottom Line: These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness.Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH.FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways.

Show MeSH
Related in: MedlinePlus