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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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Selected evolution of FPBA. (A) Aptamer sequences isolated from the first stage of SELEX. All 36 selected sequences of the 50 nt randomized region of the full length isolates comprised two families of highly related sequences. Varying residues within individual families are underlined. The first aptamer of each family was used in nitrocellulose filter-binding assays in Part B. (B) Radioactive (P32) aptamer binding to GFP immobilized on nitrocellulose demonstrates the higher affinity of G16 relative to the G3 family. (C) Aptamer sequences selected following a second stage of SELEX in which the fixed regions flanking the G16 core were optimized. About 11 other single copy sequences from a total 36 clones were not shown. The flanking sequences shared by all selected molecules are underlined. Note the shorted 3′ sequences of AP2 and AP3. (D) The nitrocellulose filter-binding assay shows the higher affinity of the selected aptamers relative to G16.
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gkr1264-F1: Selected evolution of FPBA. (A) Aptamer sequences isolated from the first stage of SELEX. All 36 selected sequences of the 50 nt randomized region of the full length isolates comprised two families of highly related sequences. Varying residues within individual families are underlined. The first aptamer of each family was used in nitrocellulose filter-binding assays in Part B. (B) Radioactive (P32) aptamer binding to GFP immobilized on nitrocellulose demonstrates the higher affinity of G16 relative to the G3 family. (C) Aptamer sequences selected following a second stage of SELEX in which the fixed regions flanking the G16 core were optimized. About 11 other single copy sequences from a total 36 clones were not shown. The flanking sequences shared by all selected molecules are underlined. Note the shorted 3′ sequences of AP2 and AP3. (D) The nitrocellulose filter-binding assay shows the higher affinity of the selected aptamers relative to G16.

Mentions: The β-barrel structure of GFP appears to have a low surface complexity, which may explain a previously unsuccessful attempt to isolate aptamers with high affinity to GFP (24). Consistent with this conjecture, initial conventional SELEX experiments with nitrocellulose as the partitioning device resulted in the progressive enrichment of membrane-binding sequences, despite repeated negative selections against membrane binding and RNaseH digestion of membrane-binding sequences (25). To overcome this problem, we alternated nitrocellulose membrane-bound GFP selection rounds with selection against His-tagged GFP bound to Ni-charged beads. Negative selection with nitrocellulose membrane or Ni-charged beads was employed at each step. This procedure resulted in strong selection pressure on the RNA pool, which consisted of two 25 nt primers flanking a 50 nt random core (Figure 1A). Following 15 iterations of binding and amplification, the sequenced RNA pool consisted of multiple copies of two highly related families, G3 and G16 (Figure 1A), both of which displayed submicromolar binding affinities in nitrocellulose filter-binding assays (Figure 1B). These species were highly enriched after 15 rounds of SELEX and showed no membrane-binding affinity. However, six other RNA species that bound to both GFP and nitrocellulose membrane, were identified at an earlier stage of SELEX (10 rounds) when G3 and G16 were enriched by >80% (Supplementary Figure S1). These results indicate that negative selection with nitrocellulose membrane eliminates RNAs that bind both membrane and GFP, thus affecting the abundance and enrichment of GFP-binding RNA species.Figure 1.


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)

Selected evolution of FPBA. (A) Aptamer sequences isolated from the first stage of SELEX. All 36 selected sequences of the 50 nt randomized region of the full length isolates comprised two families of highly related sequences. Varying residues within individual families are underlined. The first aptamer of each family was used in nitrocellulose filter-binding assays in Part B. (B) Radioactive (P32) aptamer binding to GFP immobilized on nitrocellulose demonstrates the higher affinity of G16 relative to the G3 family. (C) Aptamer sequences selected following a second stage of SELEX in which the fixed regions flanking the G16 core were optimized. About 11 other single copy sequences from a total 36 clones were not shown. The flanking sequences shared by all selected molecules are underlined. Note the shorted 3′ sequences of AP2 and AP3. (D) The nitrocellulose filter-binding assay shows the higher affinity of the selected aptamers relative to G16.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1264-F1: Selected evolution of FPBA. (A) Aptamer sequences isolated from the first stage of SELEX. All 36 selected sequences of the 50 nt randomized region of the full length isolates comprised two families of highly related sequences. Varying residues within individual families are underlined. The first aptamer of each family was used in nitrocellulose filter-binding assays in Part B. (B) Radioactive (P32) aptamer binding to GFP immobilized on nitrocellulose demonstrates the higher affinity of G16 relative to the G3 family. (C) Aptamer sequences selected following a second stage of SELEX in which the fixed regions flanking the G16 core were optimized. About 11 other single copy sequences from a total 36 clones were not shown. The flanking sequences shared by all selected molecules are underlined. Note the shorted 3′ sequences of AP2 and AP3. (D) The nitrocellulose filter-binding assay shows the higher affinity of the selected aptamers relative to G16.
Mentions: The β-barrel structure of GFP appears to have a low surface complexity, which may explain a previously unsuccessful attempt to isolate aptamers with high affinity to GFP (24). Consistent with this conjecture, initial conventional SELEX experiments with nitrocellulose as the partitioning device resulted in the progressive enrichment of membrane-binding sequences, despite repeated negative selections against membrane binding and RNaseH digestion of membrane-binding sequences (25). To overcome this problem, we alternated nitrocellulose membrane-bound GFP selection rounds with selection against His-tagged GFP bound to Ni-charged beads. Negative selection with nitrocellulose membrane or Ni-charged beads was employed at each step. This procedure resulted in strong selection pressure on the RNA pool, which consisted of two 25 nt primers flanking a 50 nt random core (Figure 1A). Following 15 iterations of binding and amplification, the sequenced RNA pool consisted of multiple copies of two highly related families, G3 and G16 (Figure 1A), both of which displayed submicromolar binding affinities in nitrocellulose filter-binding assays (Figure 1B). These species were highly enriched after 15 rounds of SELEX and showed no membrane-binding affinity. However, six other RNA species that bound to both GFP and nitrocellulose membrane, were identified at an earlier stage of SELEX (10 rounds) when G3 and G16 were enriched by >80% (Supplementary Figure S1). These results indicate that negative selection with nitrocellulose membrane eliminates RNAs that bind both membrane and GFP, thus affecting the abundance and enrichment of GFP-binding RNA species.Figure 1.

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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