Aminomodified nucleobases: functionalized nucleoside triphosphates applicable for SELEX.

Area of Science:

• Molecular Biology
• Chemical Synthesis
• Biochemistry

Background:

• The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process is crucial for developing RNA libraries with specific functionalities.
• Incorporating modified nucleoside triphosphates can introduce novel properties to RNA molecules.
• Understanding the compatibility and impact of these modifications on RNA structure and function is essential.

Purpose of the Study:

• To evaluate the suitability of three aminomodified nucleoside triphosphates for SELEX applications.
• To assess the impact of these modifications on RNA library functionality and Watson-Crick basepairing.
• To detail the chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP.

Main Methods:

• Systematic testing of 5-aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP in SELEX enzymatic steps.
• Assessment of basepairing fidelity using Watson-Crick rules.
• Measurement of melting temperatures (Tm) to determine RNA duplex stability.
• Detailed chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP.

Main Results:

• All three tested aminomodified nucleoside triphosphates are compatible with SELEX enzymatic processes and maintain Watson-Crick basepairing.
• RNA molecules modified with 5-aminoallyl-UTP and 5-aminoallyl-2'-fluoro-dUTP exhibit significantly increased melting temperatures, indicating enhanced stability.
• Incorporation of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP results in a decreased melting temperature, suggesting less stable basepairing.

Conclusions:

• Aminomodified nucleoside triphosphates can be effectively utilized in SELEX to functionalize RNA libraries.
• The choice of modification influences RNA duplex stability, with uridine analogues enhancing and the tested adenine analogue decreasing stability.
• The successful synthesis of 5-aminoallyl-2'-fluoro-dUTP provides a valuable tool for RNA engineering.