Conformational diversity versus nucleic acid triplex stability, a combinatorial study

Area of Science:

• Molecular Biology
• Biochemistry
• Oligonucleotide Chemistry

Background:

• DNA triple helix stability is crucial for applications but is poorly predictable with current modification strategies.
• Previous attempts to enhance stability involved chemical modifications of the third strand's backbone, sugar, or bases.
• A need exists for methods to rapidly screen diverse oligonucleotide conformations for improved triplex stability.

Purpose of the Study:

• To develop a method for rapid screening of conformationally diverse third strand oligonucleotides.
• To identify oligonucleotides with enhanced stability in the parallel pairing motif of DNA triple helices.
• To define basic rules governing triple helix stability in a conformationally diverse system.

Main Methods:

• Generation of combinatorial oligonucleotide libraries varying in sugar units (ribose/deoxyribose) at each position.
• Affinity chromatography using immobilized DNA target duplexes.
• Temperature gradient selection to identify stable triple helix-forming oligonucleotides, followed by physicochemical characterization.

Main Results:

• Identified oligonucleotides that form stable triple helices, enabling the definition of stability rules.
• Ribocytidines in the third strand significantly increase triplex stability compared to deoxyribocytidines.
• Stability of (deoxy)thymidines and uridines in the third strand is highly dependent on the specific sequence.

Conclusions:

• The developed screening method allows for efficient identification of stable third strand oligonucleotides.
• Incorporation of ribocytidines is a general strategy to enhance DNA triple helix stability.
• Sequence context plays a critical role in the stability contribution of (deoxy)thymidines and uridines.