A Base Pair Outside the Catalytic Core of the I-R3 DNA Enzyme Has a Significant Effect on Its Cleavage Activity: An Improved Catalytic Core Model and an Automated Design Program
The I-R3 DNA enzyme, in its trans-acting form, is capable of cleaving single-stranded DNA (ssDNA) molecules. We have collected all published information on the activity levels of the original I-R3 DNA enzyme and its known variants and embedded that information into a program (we called IR3). The pro...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Wiley
2025-01-01
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| Series: | Journal of Nucleic Acids |
| Online Access: | http://dx.doi.org/10.1155/jna/5518018 |
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| Summary: | The I-R3 DNA enzyme, in its trans-acting form, is capable of cleaving single-stranded DNA (ssDNA) molecules. We have collected all published information on the activity levels of the original I-R3 DNA enzyme and its known variants and embedded that information into a program (we called IR3). The program was applied to the sequences of a set of ssDNA viruses and identified all potential catalytic core substrates (targets) and output optimal I-R3 DNA enzyme sequences for all the targets, along with expected activity levels of the enzymes at those targets. Upon experimentally measuring the in vitro cleavage activities of the I-R3 variants, we found marked differences between the program-predicted and experimentally measured values. This demonstrated the incompleteness of the I-R3 model: The sequence of the nucleotides of the catalytic core is not sufficient to fully determine its activity level. A set of experiments was carried out in which the effect of all possible combinations of Watson–Crick base pairs at two positions near the catalytic core, termed SI and SII, was tested. To confirm a newly formed hypothesis, the nucleotide at the SII position of the enzyme strand was mutated to a G and a T, with the substrate strand mutated accordingly. In every case, this led to an increase in relative activity when changed to a G and a decrease, when changed to a T, of the variant I-R3 DNA enzyme. Clearly, the discovered base pair peripheral to the catalytic core has a substantial effect on cleavage activity. This improves the current model of essential nucleotides, and the IR3 software outputs I-R3 enzyme-sequence recommendations that make them more likely to cleave their targets. The software is available for download at https://github.com/XinxinTree/IR3. |
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| ISSN: | 2090-021X |