Have you noticed, formation and dissociation of triplex DNA is not fully reversible upon pH titration? After several cycles it becomes almost irreversible, i.e. fold in triplex conformation. Any thoughts?
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Antonio S Minero ,
The reversible formation and dissociation of triplex DNA can be influenced by various factors, including pH changes. Triplex DNA typically involves the formation of a third DNA strand (the triplex-forming oligonucleotide or TFO) binding to the major groove of a double-stranded DNA (dsDNA) molecule through Hoogsteen base pairing. The stability of this triplex structure can be affected by several factors:
1. **pH:** The pH of the solution can have a significant impact on the stability of triplex DNA. At different pH levels, the protonation states of the DNA bases can change, which, in turn, can affect the binding and stability of the triplex structure.
2. **Ionic Strength:** The concentration of ions in the solution, particularly divalent cations like Mg²⁺, can influence the stability of triplex DNA. These ions can help stabilize the triplex structure through electrostatic interactions.
3. **Temperature:** Triplex formation is temperature-dependent. Lower temperatures generally favor triplex formation, while higher temperatures can promote dissociation.
4. **Sequence Specificity:** The sequence of the TFO and the target dsDNA plays a crucial role in triplex stability. Specific sequences may form more stable triplex structures than others.
5. **Structural Features:** The presence of mismatches, bulges, or other structural irregularities in the TFO or the target dsDNA can affect the stability of the triplex.
6. **Base Modifications:** Chemical modifications to the DNA bases, such as methylation or incorporation of analogs, can alter the stability of the triplex.
It's not uncommon for DNA structures to exhibit hysteresis in their stability during pH titration or other environmental changes. This hysteresis can be due to several reasons:
1. **Kinetic Trapping:** Sometimes, the transition between different DNA conformations (like triplex to duplex) may involve kinetic barriers. Even if the conditions are reversed, the system might not readily return to its initial state due to these barriers.
2. **Structural Changes:** pH changes can induce structural changes in the DNA or alter the protonation states of specific bases. These changes may not fully revert when the pH is reversed.
3. **Aggregation:** DNA molecules can aggregate or interact with other molecules or surfaces under certain conditions, making it difficult for them to return to their original state.
4. **Chemical Modifications:** pH changes can lead to chemical modifications of DNA bases, which can be irreversible.
To address the issue of irreversibility in triplex formation and dissociation, you might consider careful optimization of experimental conditions, such as pH, temperature, and ion concentrations. Additionally, studying the kinetics of triplex formation and dissociation can provide insights into the underlying processes and help identify ways to mitigate irreversibility. It's important as well to be aware that the specific DNA sequences and environmental conditions can greatly influence the behavior of triplex DNA.
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