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Updated: Jun 24, 2026
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Effective DNA inhibitors of cathepsin g by in vitro selection
Barbara Gatto1, Elena Vianini1, Lorena Lucatello1
1Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, 35131 Padova, Italy.
Insights
Short DNA sequences, particularly those with alternating TG repeats, effectively inhibit Cathepsin G (CatG). These findings highlight DNA
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cathepsin G (CatG), a neutrophil protease, contributes to tissue damage and platelet aggregation in inflammatory and ischemic diseases.
- An imbalance between CatG and its inhibitors exacerbates disease pathology.
- DNA released from dying neutrophils can inhibit CatG, with short fragments being more potent than genomic DNA.
Purpose of the Study:
- To identify specific single-stranded DNA sequences with high affinity for Cathepsin G.
- To investigate the structural motif responsible for DNA-mediated CatG inhibition.
- To explore the therapeutic potential of DNA-based CatG inhibitors.
Main Methods:
- A SELEX (Systematic Evolution of Ligands by Exponential Enrichment) protocol was employed to select high-affinity DNA sequences for CatG.
- Biophysical and biochemical assays were used to characterize the binding and inhibitory activity of selected DNA sequences.
- Sequence analysis was performed to identify recurring motifs responsible for CatG recognition.
Main Results:
- Specific single-stranded DNA sequences were identified that bind tightly to CatG and efficiently inhibit its catalytic activity.
- Alternating TG repeats were found to be a recurring motif in the high-affinity DNA binders.
- Selected DNA sequences, designed to minimize folding, demonstrated potent CatG inhibition.
Conclusions:
- Specific DNA sequences, characterized by alternating TG repeats and an extended conformation, are potent inhibitors of Cathepsin G.
- These findings provide a molecular basis for DNA-mediated CatG inhibition and suggest potential therapeutic strategies for inflammatory and procoagulant conditions.
- Defibrotide's inhibitory effect on CatG may be attributed to its polydeoxyribonucleotide nature and specific sequence composition.
Abstract:
Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions.
