Ultra-Stable RNA-Based Monomolecular Triplexes

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Stable nucleic acid triplexes form via Hoogsteen interactions between a third strand (TFO) and a homopurine:homopyrimidine duplex.
• Conventional triplexes use U-shaped loops and TFOs do not typically enhance duplex stability.
• Noncanonical quadruplexes utilize Z-shaped loops for high stability.

Purpose of the Study:

• To investigate if Z-shaped loops can increase triplex stability.
• To design and characterize novel DNA and RNA triplexes with mixed loop types (U-shaped and Z-shaped).
• To determine the effect of these novel triplexes on the stability of underlying DNA and RNA duplexes.

Main Methods:

• Design and synthesis of novel oligonucleotide sequences incorporating Z-shaped loops.
• Formation and characterization of intramolecular DNA and RNA triplexes.
• Thermal denaturation studies (melting point analysis) to assess duplex and triplex stability.

Main Results:

• A novel folding principle was demonstrated using two adjacent GGG segments with an intervening nucleotide forming a Z-shaped loop.
• Designed DNA and RNA triplexes with one U-shaped and one Z-shaped loop were successfully created.
• The TFO enhanced the stability of underlying RNA duplexes but not DNA duplexes.
• Stabilized triplexes unfolded cooperatively above the melting point of the RNA duplex.

Conclusions:

• Ultrastable Z-looped intramolecular triplexes can form in vitro.
• This structural motif enhances RNA duplex stability, unlike conventional triplexes.
• These findings reveal a new structural motif with potential as a therapeutic target against pathogenic RNAs.