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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

FPBAs alter protein fluorescence. (A) Emission scan (475 nm excitation) of GFP in the presence of increasing concentrations of AP3. Inset shows dose-dependent inhibition of fluorescence at 505 nm; GFP concentration was 25 nM. No aptamers displayed measurable fluorescence and the original unselected aptamer pool did not alter GFP fluorescence (not shown). (B) Inhibition of GFP fluorescence by G16 and AP3 (475/505), showing higher affinity and greater maximum effect of AP3. (C) Fluorescence emission scans of enhanced FPs show the distinct functional effects of AP3. All spectra are normalized to peak fluorescence in the absence of AP3 and reflect 10 nM of FP and 250 nM AP3. Note increase in ECFP and EYFP fluorescence in the presence of AP3.
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gkr1264-F3: FPBAs alter protein fluorescence. (A) Emission scan (475 nm excitation) of GFP in the presence of increasing concentrations of AP3. Inset shows dose-dependent inhibition of fluorescence at 505 nm; GFP concentration was 25 nM. No aptamers displayed measurable fluorescence and the original unselected aptamer pool did not alter GFP fluorescence (not shown). (B) Inhibition of GFP fluorescence by G16 and AP3 (475/505), showing higher affinity and greater maximum effect of AP3. (C) Fluorescence emission scans of enhanced FPs show the distinct functional effects of AP3. All spectra are normalized to peak fluorescence in the absence of AP3 and reflect 10 nM of FP and 250 nM AP3. Note increase in ECFP and EYFP fluorescence in the presence of AP3.

Mentions: Fluorescence emission spectra were collected using either a FluoroMax-3 spectrofluorometer (JY Horiba, Edison, NJ, USA) or a QuantaMaster 40 (PTI, Birmingham, NJ, USA). Aptamer and fluorescence proteins were prepared in buffer 1: 1× PBS (pH 7.4) with 5 mM MgCl2 (Figures 3 and 4A and B), or buffer 2: 120 mM KCl, 5 mM NaCl, 1 mM MgCl, 10 mM MES, 10 mM MOPS and 10 mM citrate (pH titration experiments). Fluorescence intensities were measured by exciting at 475 nm for GFP and EGFP, 453 nm for ECFP and 513 nm for EYFP. Absorbance measurements were carried out using a Cary 300 dual beam spectrophotometer (Varian, Palo Alto, CA, USA). Data from the EGFP pH titration experiments were fit to A + B/(1 – 10(nH(pKa-pH))) where nH is the Hill coefficient (21).


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)

FPBAs alter protein fluorescence. (A) Emission scan (475 nm excitation) of GFP in the presence of increasing concentrations of AP3. Inset shows dose-dependent inhibition of fluorescence at 505 nm; GFP concentration was 25 nM. No aptamers displayed measurable fluorescence and the original unselected aptamer pool did not alter GFP fluorescence (not shown). (B) Inhibition of GFP fluorescence by G16 and AP3 (475/505), showing higher affinity and greater maximum effect of AP3. (C) Fluorescence emission scans of enhanced FPs show the distinct functional effects of AP3. All spectra are normalized to peak fluorescence in the absence of AP3 and reflect 10 nM of FP and 250 nM AP3. Note increase in ECFP and EYFP fluorescence in the presence of AP3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1264-F3: FPBAs alter protein fluorescence. (A) Emission scan (475 nm excitation) of GFP in the presence of increasing concentrations of AP3. Inset shows dose-dependent inhibition of fluorescence at 505 nm; GFP concentration was 25 nM. No aptamers displayed measurable fluorescence and the original unselected aptamer pool did not alter GFP fluorescence (not shown). (B) Inhibition of GFP fluorescence by G16 and AP3 (475/505), showing higher affinity and greater maximum effect of AP3. (C) Fluorescence emission scans of enhanced FPs show the distinct functional effects of AP3. All spectra are normalized to peak fluorescence in the absence of AP3 and reflect 10 nM of FP and 250 nM AP3. Note increase in ECFP and EYFP fluorescence in the presence of AP3.
Mentions: Fluorescence emission spectra were collected using either a FluoroMax-3 spectrofluorometer (JY Horiba, Edison, NJ, USA) or a QuantaMaster 40 (PTI, Birmingham, NJ, USA). Aptamer and fluorescence proteins were prepared in buffer 1: 1× PBS (pH 7.4) with 5 mM MgCl2 (Figures 3 and 4A and B), or buffer 2: 120 mM KCl, 5 mM NaCl, 1 mM MgCl, 10 mM MES, 10 mM MOPS and 10 mM citrate (pH titration experiments). Fluorescence intensities were measured by exciting at 475 nm for GFP and EGFP, 453 nm for ECFP and 513 nm for EYFP. Absorbance measurements were carried out using a Cary 300 dual beam spectrophotometer (Varian, Palo Alto, CA, USA). Data from the EGFP pH titration experiments were fit to A + B/(1 – 10(nH(pKa-pH))) where nH is the Hill coefficient (21).

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