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Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing.

Huang PJ, Liu J - Nucleic Acids Res. (2015)

Bottom Line: A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd(2+) due to limited functional groups in DNA.Its application in detecting Cd(2+) is also demonstrated.The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.

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(A) A scheme for the blocked selection and with negative selection. The blockers are intended to eliminate the Ce13 sequences. Steps 1, 2 and 3 consist of a completely blocked selection cycle. From round 8, negative selections were carried out with a metal mixture. In this case, the uncleaved oligonucleotides were collected (steps 4, 5 and 6) and then reacted with Cd2+ for the positive selection (steps 2 and 3). (B) Selection progress from round 8 of the blocked selection. For each round, both positive and negative selections were carried out. The round 15 library was sequenced. (C) A trans-cleaving DNAzyme derived from BN-Cd16. (D) Alignment of the enzyme loop for sequences similar to BN-Cd16. Nucleotides in red are absolutely conserved, in blue can be purine or pyrimidine substituted and in yellow are variable. The clone numbers are in the parentheses. The color coding matches that in (C).
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Figure 2: (A) A scheme for the blocked selection and with negative selection. The blockers are intended to eliminate the Ce13 sequences. Steps 1, 2 and 3 consist of a completely blocked selection cycle. From round 8, negative selections were carried out with a metal mixture. In this case, the uncleaved oligonucleotides were collected (steps 4, 5 and 6) and then reacted with Cd2+ for the positive selection (steps 2 and 3). (B) Selection progress from round 8 of the blocked selection. For each round, both positive and negative selections were carried out. The round 15 library was sequenced. (C) A trans-cleaving DNAzyme derived from BN-Cd16. (D) Alignment of the enzyme loop for sequences similar to BN-Cd16. Nucleotides in red are absolutely conserved, in blue can be purine or pyrimidine substituted and in yellow are variable. The clone numbers are in the parentheses. The color coding matches that in (C).

Mentions: To isolate new DNAzymes, we decided to re-select. A few methods (e.g. mutagenic PCR) might increase sequence diversity and reduce the Ce13 sequence. Here we developed a new method based on the property of Ce13. We noticed that the conserved sequence of Ce13 is quite long (e.g. the 15 nucleotides in cyan in Figure 1C). Only one of the nucleotides (marked by underline) may change from G to A (22). We hypothesized that the library might be evolved against this sequence by using blocking DNA complementary to these conserved nucleotides. Two blocking DNAs were designed in Figure 1C, and they can completely inactivate the Ce13 DNAzyme (Figure 1D). In our new selection scheme (Figure 2A), an excess amount of blocking DNAs (150 pmol) was first hybridized with the library to inactivate the Ce13 sequences (step 1). Then Cd2+ was added (step 2) and the cleaved oligonucleotides were amplified (step 3). The progress was slightly slower and it took seven rounds to reach activity plateau (Figure 1B, gray bars). This is probably due to suppression of the highly active Ce13 population. The round 7 library was sequenced and the Ce13 variants were indeed eliminated (see Supplementary Table S5 for sequence alignment). This enriched library however has a high sequence diversity, suggesting that many solutions for Cd2+-dependent cleavage are available. After treating the round 7 library with a metal mixture (Pb2+, Cu2+ and Zn2+, 20 μM each), nearly 60% cleavage occurred after 1 h, indicating this library still lacked specificity for Cd2+.


Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing.

Huang PJ, Liu J - Nucleic Acids Res. (2015)

(A) A scheme for the blocked selection and with negative selection. The blockers are intended to eliminate the Ce13 sequences. Steps 1, 2 and 3 consist of a completely blocked selection cycle. From round 8, negative selections were carried out with a metal mixture. In this case, the uncleaved oligonucleotides were collected (steps 4, 5 and 6) and then reacted with Cd2+ for the positive selection (steps 2 and 3). (B) Selection progress from round 8 of the blocked selection. For each round, both positive and negative selections were carried out. The round 15 library was sequenced. (C) A trans-cleaving DNAzyme derived from BN-Cd16. (D) Alignment of the enzyme loop for sequences similar to BN-Cd16. Nucleotides in red are absolutely conserved, in blue can be purine or pyrimidine substituted and in yellow are variable. The clone numbers are in the parentheses. The color coding matches that in (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499143&req=5

Figure 2: (A) A scheme for the blocked selection and with negative selection. The blockers are intended to eliminate the Ce13 sequences. Steps 1, 2 and 3 consist of a completely blocked selection cycle. From round 8, negative selections were carried out with a metal mixture. In this case, the uncleaved oligonucleotides were collected (steps 4, 5 and 6) and then reacted with Cd2+ for the positive selection (steps 2 and 3). (B) Selection progress from round 8 of the blocked selection. For each round, both positive and negative selections were carried out. The round 15 library was sequenced. (C) A trans-cleaving DNAzyme derived from BN-Cd16. (D) Alignment of the enzyme loop for sequences similar to BN-Cd16. Nucleotides in red are absolutely conserved, in blue can be purine or pyrimidine substituted and in yellow are variable. The clone numbers are in the parentheses. The color coding matches that in (C).
Mentions: To isolate new DNAzymes, we decided to re-select. A few methods (e.g. mutagenic PCR) might increase sequence diversity and reduce the Ce13 sequence. Here we developed a new method based on the property of Ce13. We noticed that the conserved sequence of Ce13 is quite long (e.g. the 15 nucleotides in cyan in Figure 1C). Only one of the nucleotides (marked by underline) may change from G to A (22). We hypothesized that the library might be evolved against this sequence by using blocking DNA complementary to these conserved nucleotides. Two blocking DNAs were designed in Figure 1C, and they can completely inactivate the Ce13 DNAzyme (Figure 1D). In our new selection scheme (Figure 2A), an excess amount of blocking DNAs (150 pmol) was first hybridized with the library to inactivate the Ce13 sequences (step 1). Then Cd2+ was added (step 2) and the cleaved oligonucleotides were amplified (step 3). The progress was slightly slower and it took seven rounds to reach activity plateau (Figure 1B, gray bars). This is probably due to suppression of the highly active Ce13 population. The round 7 library was sequenced and the Ce13 variants were indeed eliminated (see Supplementary Table S5 for sequence alignment). This enriched library however has a high sequence diversity, suggesting that many solutions for Cd2+-dependent cleavage are available. After treating the round 7 library with a metal mixture (Pb2+, Cu2+ and Zn2+, 20 μM each), nearly 60% cleavage occurred after 1 h, indicating this library still lacked specificity for Cd2+.

Bottom Line: A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd(2+) due to limited functional groups in DNA.Its application in detecting Cd(2+) is also demonstrated.The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.

Show MeSH
Related in: MedlinePlus