[New Method for Evaluating the Substrate Efficiency of Modified Deoxynucleoside Triphosphates for Selex]

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Modified aptamers offer enhanced functionality for various applications.
• The selection of modified aptamers (mod-SELEX) requires specialized deoxynucleoside triphosphates (dNTPs).
• Evaluating the suitability of modified dNTPs for mod-SELEX is crucial but can be time-consuming.

Purpose of the Study:

• To develop a rapid method for assessing the substrate efficiency of functionalized dNTPs for mod-SELEX.
• To provide a tool for determining the compatibility of modified dNTPs with specific PCR conditions and polymerases.
• To facilitate the selection of aptamers for clinical diagnostics, medicine, and scientific research.

Main Methods:

• A three-round real-time PCR amplification process using a combinatorial library and a candidate modified dNTP.
• Monitoring the amplification curve's changes over consecutive PCR rounds to assess library representation.
• Evaluating dNTP derivative suitability based on amplification curve stability, indicating no competitive amplification or library degeneration.

Main Results:

• The method allows for real-time assessment of modified dNTP substrate efficiency without requiring full SELEX rounds.
• Degeneration of the amplification curve indicates that a modified dNTP is incompatible with the chosen polymerase and amplification conditions.
• Stable amplification curves suggest that the modified dNTP does not alter the library's sequence distribution, making it suitable for mod-SELEX.

Conclusions:

• The proposed method offers a quick and effective way to evaluate modified dNTPs for mod-SELEX.
• This approach streamlines the process of identifying suitable modified dNTPs for aptamer selection.
• The findings will aid in the development of novel aptamers for diverse research and clinical applications.