Specific RNA self-assembly with minimal paranemic motifs

Area of Science:

• * Molecular Biology
• * RNA Nanotechnology
• * Biophysics

Background:

• * Paranemic crossover (PX) enables nucleic acid assembly via Watson-Crick (WC) basepairing without unfolding secondary structures.
• * Previous PX assembly demonstrated in DNA, but not RNA, requiring ≥4 crossovers and ≥5 helical half-turns.
• * Reversible nature of PX assembly avoids denaturation of internal basepairs.

Purpose of the Study:

• * To design RNA molecules capable of paranemic assembly.
• * To create minimal RNA paranemic motifs with two crossovers and three helical half-turns (3HT).
• * To characterize the stability and kinetics of these novel RNA assemblies.

Main Methods:

• * Design and synthesis of RNA molecules with specific crossover points.
• * Measurement of dissociation constants (Kd) to assess complex stability.
• * Determination of kinetic exchange half-times to calculate association (ka) and dissociation (kd) rate constants.
• * Mutational analysis to investigate the role of WC basepairing in assembly.

Main Results:

• * Successful design of RNA molecules forming 3HT paranemic motifs with two crossovers.
• * Identification of the 3HT complex with six basepairs (3HT_6M) as the most stable (Kd = 1 x 10-8 M).
• * 3HT_6M complex exhibited a long kinetic exchange half-time (~100 min), indicating high stability (ka = 5.11 x 103 M-1s-1, kd = 5.11 x 10-5 s-1).
• * Assembly was sensitive to single-base substitutions disrupting WC basepairs and restored by compensatory substitutions.

Conclusions:

• * Demonstrated RNA paranemic assembly with minimal crossovers (2) and length (3HT).
• * The 3HT_6M motif shows significant stability and slow dissociation, suitable for reversible self-assembly.
• * This work provides a foundation for programmable tecto-RNA self-assembly and the construction of artificial RNA molecular machines.