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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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Confirmed secondary structure of AP3. (A) Schematic diagram of the secondary structure of AP3 (143 nt). Stem–loop 1 was formed by the core sequence from G16. (B) Secondary structure of minimized AP3 (83 nt). Stem–loop 2 and Stem–loop 3 were shortened by truncating from the apex loops of both Stem–loops inward. New random loops were added to the apexes of both minimized stems. The circle indicates site where addition of paired nucleotides between positions 20/21 and 79/80 does not alter binding to GFP, whereas addition of single nucleotide at either site resulted in loss of binding, confirming the stem structure in this region. Insertion of nucleotides between Stem–loop 1 and Stem–loop 2 (arrow 1) also reduced binding to GFP. Dotted lines show other 5′-/3′-ends of fully functional circular permutated aptamers. (C) Re-selection of Stem–loop 3 resulted in reduction of the internal loop (G marked by asterisk in A and B for orientation). Removal of nucleotides within this loop, even a single nucleotide deletion from either the 5′- or 3′-end in the form shown in (A), resulted in loss of function.
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gkr1264-F5: Confirmed secondary structure of AP3. (A) Schematic diagram of the secondary structure of AP3 (143 nt). Stem–loop 1 was formed by the core sequence from G16. (B) Secondary structure of minimized AP3 (83 nt). Stem–loop 2 and Stem–loop 3 were shortened by truncating from the apex loops of both Stem–loops inward. New random loops were added to the apexes of both minimized stems. The circle indicates site where addition of paired nucleotides between positions 20/21 and 79/80 does not alter binding to GFP, whereas addition of single nucleotide at either site resulted in loss of binding, confirming the stem structure in this region. Insertion of nucleotides between Stem–loop 1 and Stem–loop 2 (arrow 1) also reduced binding to GFP. Dotted lines show other 5′-/3′-ends of fully functional circular permutated aptamers. (C) Re-selection of Stem–loop 3 resulted in reduction of the internal loop (G marked by asterisk in A and B for orientation). Removal of nucleotides within this loop, even a single nucleotide deletion from either the 5′- or 3′-end in the form shown in (A), resulted in loss of function.

Mentions: To confirm the secondary structure and optimize Stem–loop 3 of AP3, a circular permutated RNA pool was generated by introducing 5′-/3′-ends to the hairpin loop of Stem–loop 2 and randomizing the intact Stem–loop 3 and flank junction loop (see the sequences 1–25 and 76–83 in Figure 5B for coordinates). A selection was performed according to the procedure described above.


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)

Confirmed secondary structure of AP3. (A) Schematic diagram of the secondary structure of AP3 (143 nt). Stem–loop 1 was formed by the core sequence from G16. (B) Secondary structure of minimized AP3 (83 nt). Stem–loop 2 and Stem–loop 3 were shortened by truncating from the apex loops of both Stem–loops inward. New random loops were added to the apexes of both minimized stems. The circle indicates site where addition of paired nucleotides between positions 20/21 and 79/80 does not alter binding to GFP, whereas addition of single nucleotide at either site resulted in loss of binding, confirming the stem structure in this region. Insertion of nucleotides between Stem–loop 1 and Stem–loop 2 (arrow 1) also reduced binding to GFP. Dotted lines show other 5′-/3′-ends of fully functional circular permutated aptamers. (C) Re-selection of Stem–loop 3 resulted in reduction of the internal loop (G marked by asterisk in A and B for orientation). Removal of nucleotides within this loop, even a single nucleotide deletion from either the 5′- or 3′-end in the form shown in (A), resulted in loss of function.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1264-F5: Confirmed secondary structure of AP3. (A) Schematic diagram of the secondary structure of AP3 (143 nt). Stem–loop 1 was formed by the core sequence from G16. (B) Secondary structure of minimized AP3 (83 nt). Stem–loop 2 and Stem–loop 3 were shortened by truncating from the apex loops of both Stem–loops inward. New random loops were added to the apexes of both minimized stems. The circle indicates site where addition of paired nucleotides between positions 20/21 and 79/80 does not alter binding to GFP, whereas addition of single nucleotide at either site resulted in loss of binding, confirming the stem structure in this region. Insertion of nucleotides between Stem–loop 1 and Stem–loop 2 (arrow 1) also reduced binding to GFP. Dotted lines show other 5′-/3′-ends of fully functional circular permutated aptamers. (C) Re-selection of Stem–loop 3 resulted in reduction of the internal loop (G marked by asterisk in A and B for orientation). Removal of nucleotides within this loop, even a single nucleotide deletion from either the 5′- or 3′-end in the form shown in (A), resulted in loss of function.
Mentions: To confirm the secondary structure and optimize Stem–loop 3 of AP3, a circular permutated RNA pool was generated by introducing 5′-/3′-ends to the hairpin loop of Stem–loop 2 and randomizing the intact Stem–loop 3 and flank junction loop (see the sequences 1–25 and 76–83 in Figure 5B for coordinates). A selection was performed according to the procedure described above.

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