In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing.
Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na(+)), has remained underdeveloped, even though Na(+) is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na(+)-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (ko(bs)) ∼ 0.1 min(-1)], and the transformation of this DNAzyme into a fluorescent sensor for Na(+) by labeling the enzyme strand with a quencher at the 3' end, and the DNA substrate strand with a fluorophore and a quencher at the 5' and 3' ends, respectively. The presence of Na(+) catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na(+) over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na(+)-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na(+) in living cells.
Departments of Biochemistry.
Seyed-Fakhreddin
SF
Torabi
Departments of Biochemistry.
Peiwen
P
Wu
Chemistry, and.
Claire E
CE
McGhee
Departments of Biochemistry.
Lu
L
Chen
Chemistry, and.
Kevin
K
Hwang
Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Nan
N
Zheng
Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Jianjun
J
Cheng
Departments of Biochemistry, Chemistry, and Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 yi-lu@illinois.edu.
Yi
Y
Lu
D015374:Biosensing Techniques; D002384:Catalysis; D002412:Cations; D021881:DNA, Catalytic / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007477:Ions; D008670:Metals / Q000737:chemistry; D018613:Microscopy, Confocal; D009696:Nucleic Acids / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry; D012964:Sodium / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D002412:Cations; D021881:DNA, Catalytic; D005456:Fluorescent Dyes; D007477:Ions; D008670:Metals; D009696:Nucleic Acids; D010455:Peptides; D012313:RNA; D012964:Sodium; D011188:Potassium
deoxyribozyme; fluorescent sensor; in vitro selection; intracellular imaging; sodium