Team:OSA/DNAzyme Mutation

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Objective

1.Through mutation test, we hope to find a 8-17 DNAzyme mutant with higher activity and sensitivity.

2.We also hope to determine the relationship between 8-17 DNAzyme's sequence and its activity.

Procedures:

We modified the large or small stem ring of the original 8-17 DNAzyme. There are 95 mutants in total.

Fig. 2.4.1 2D structure of 8-17 DNAzyme

Semi-rational mutation design:

For mutant 1-9, we replaced a single base pair on their large stem ring.

For mutant 10-24, we replaced a single base pair on their small stem ring.

For mutant 25-40, we inserted a single base pair into their large stem ring.

For mutant 41-64, we inserted a single base pair into their large stem ring.

For mutant 65-67, we reversed the base pairs on their small stem ring.

For mutant 68-70, we modified the base pairs on their small stem ring.

For mutant 71-79, we modified the base pairs on the end of their stem rings.

For mutant 80-83, we elongated the base pairs on their large stem ring.

For mutant 84-87, we elongated the base pairs on their small stem ring.

For mutant 88-95, we elongated the base pairs on their stem rings.

2.We conducted preliminary test on the 95 mutants and selected 14 mutants that might be effective.

Materials:

10 μM Pb2+, 500 nM 8-17 DNAzyme, 500 nM substrate strand, 500 nM target.

Fig. 2.4.2 8-17 DNAzyme mutation test

Selected 14 mutants:

mut 9, mut 10, mut 11, mut 12, mut 20, mut 21, mut 23, mut 24, mut 35, mut 39, mut 60, mut 64, mut 65, mut 70.

3.We tested the 14 mutants in order to determine the most effective one. The testing was repeated five times.

Most effective mutant: mut 10

4.We compared the detection limit of the original DNAzyme and mut 10.

Fig. 2.4.3 DNAzyme dectection test. Left: mut 10; Right: Original

Conclusion:

Through the mutation test, we discovered that mut 10 possesses higher activity and approximately same sensitivity as the original 8-17 DNAzyme.