A new heavy lanthanide-dependent DNAzyme displaying strong metal cooperativity and unrescuable phosphorothioate effect

Abstract

In vitro selection of RNA-cleaving DNAzymes was performed using three heavy lanthanide ions (Ln3+): Ho3+, Er3+ and Tm3+. The resulting sequences were aligned together and about half of the library contained a new family of DNAzyme. These DNAzymes have a simple loop structure, and they are active only with the seven heavy Ln3+. Among the tested non-lanthanide ions, only Y3+ induced cleavage and even Pb2+ failed to cleave, suggesting a very high specificity. A representative DNAzyme, Tm7, has a sigmoidal metal binding curve with a Hill coefficient of 3, indicating that three metal ions are involved in the catalytic step. Its pH-rate profile has a slope of 1, suggesting a single deprotonation step is involved in the rate-limiting step. Tm7 has a cleavage rate of 1.6 min−1 at pH 7.8 with 10 μM Er3+. Phosphorothioate substitution at the cleavage junction completely inhibits the activity, which cannot be rescued by Cd2+ alone, or by a mixture of Er3+ and Cd2+, suggesting that two interacting metal ions are involved in direct bonding to both non-bridging oxygen atoms. A new model involving three lanthanide ions is proposed based on this study. A biosensor is engineered using Tm7 to detect Dy3+ down to 14 nM.

In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage

“…42 We recently reported that the Tm7 DNAzyme employs three Ln 3+ ions cooperatively. 30 In this study, Dy10a employs two Ln 3+ ions, further supporting the assertion that it is a new DNAzyme. Dy10a is also a very efficient enzyme; its rate reaches ~0.7 min -1 with 0.5 µM Sm 3+ , the highest among all reported Ln 3+ -dependent DNAzymes at pH 6.…”

“…Tm7 is nearly inactive with the first seven light Ln 3+ but is highly active with the heavy ones. 30 Tm7 binds three Ln 3+ ions cooperatively, which has never been observed previously for DNAzymes.…”

“…We also converted Dy35 into a trans-cleaving form and it is also active (the last lane in Figure 1D). Since Dy10a appears to be different from all the previously reported Ln 3+ -dependent DNAzymes, [28][29][30] we characterized it in detail. With only two active sequences, the information from the DNA library is limited, only supporting the overall secondary structure and two mutable nucleotides.…”

“…Y 3+ has a size similar to Ho 3+ and it can activate the previously reported DNAzymes. [28][29][30] Interestingly, 5 µM Pb 2+ did not show any cleavage. Based on previous studies, Pb 2+ is active with many DNAzymes.…”

“…For comparison, the Tm7 DNAzyme binds three lanthanide ions and it is completely inactive with the PS modification, regardless of the Rp or Sp form, and regardless of the type of metal ion tested. 30 On the other hand, most DNAzymes/ribozymes binding one metal have partially retained activity with the PS substrate and the isomer can often be fully rescued by Cd 2+ . Based on the above biochemical studies, we proposed a metal binding scheme in Figure 5C.…”