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As a result, two strands of unequal length were produced after PCR2, allowing isolation of the positive strand containing rA via gel electrophoresis to seed the next round of selection. P4 has a polymer spacer that can stop the polymerase reaction. P3 has a FAM label on its 5′-end and a ribo-adenosine (rA) base near its 3′-end. In PCR1, the full-length library was regenerated, and in PCR2, two special primers were used. Two rounds of polymerase chain reactions (PCR) were carried out to amplify the cleaved DNA (primer sequences in Table S1, Supporting Information). After incubating the library with the salt, the cleaved sequences were isolated using gel electrophoresis since they were 28-nucleotide shorter than the original full-length DNA. (NH 4) 2Ce(NO 3) 6 was used as the Ce 4+ source because of its excellent solubility and stability. (44) Our initial library contained ∼10 14 random DNA sequences. There is a single RNA linkage (rA) in the whole sequence to provide a scissile bond, since RNA is about one million fold less stable than DNA. The in vitro selection experiment was carried out using a DNA library containing a 50-nucleotide randomized region (N 50) as shown in Figure 1A (in blue). Dried DNA was resuspended in 70 μL of 5 mM HEPES buffer (pH 7.5). The purified selected DNA was then dried in an Eppendorf Vacufuge at 30 ☌ overnight. A fraction of the selected DNA was extracted from the gel and further purified with a Sep-Pak C18 column (Waters). After incubation, the reaction was quenched with 8 M urea and purified in 10% dPAGE. Incubation time and concentration of metal salt are in Table S2, Supporting Information. For the in vitro selection experiment, the random DNA pool was incubated with freshly prepared Ce(IV) metal ion. For each of the subsequent rounds the library was generated from PCR. This DNA was used directly as the DNA library for the first round of selection. The extracted DNA library was further concentrated via ethanol precipitation and resuspended in 60 μL of buffer C (50 mM MES, pH 6.0, 25 mM NaCl), which was the selection buffer. Ligated DNA product was purified with 10% denaturing polyacrylamide gel (dPAGE) at 650 V for 1 h, and DNA was extracted from the gel with buffer B (1 mM EDTA, 10 mM Tris-HCl, pH 7.0). The T4 ligation protocol provided by New England Biolabs was followed for the ligation reaction. The three strands of DNA were annealed at 95 ☌ for 1 min followed by slow cooling to room temperature. Lib-FAM DNA (200 pmol) and Lib-rA DNA (300 pmol) were mixed with splint DNA (300 pmol) first in buffer A (50 mM pH 7.5 Tris-HCl, pH 7.5, 10 mM MgCl 2). Out of the many biomolecules, DNAzymes have emerged as a unique platform for designing metal biosensors.įor this in vitro selection experiment the initial DNA library was prepared by ligating two pieces of DNA (Lib-FAM and Lib-rA) with a splint DNA. While lanthanide complexes have been used as a label for immunoassays, using the biosensor strategy for their detection has not been explored. At the same time, no general sensors are available to detect all the lanthanides with the same sensitivity either. In addition, the current sensors detect a few lanthanides no sensor can selectively detect each element. (4, 5) Most of these small organic molecule-based probes require organic solvents with poor sensitivity. A number of ion-selective electrodes and fluorescent/colorimetic chelators have been reported for lanthanides. To this end, sensors provide a useful alternative. It is more desirable to perform on-site and real-time detection. (3) While these methods provide high sensitivity and detect a few metals at the same time, they often require multiple steps of sample pretreatment and sending samples to centralized laboratories at a high cost with a long turnaround time. Analysis of lanthanides is currently carried out using instrumentation methods such as inductively coupled plasma-mass spectrometry (ICP-MS), capillary electrophoresis, and vibration spectroscopy.
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