A complex RNA-cleaving DNAzyme that can efficiently cleave a pyrimidine-pyrimidine junction

Area of Science:

• Biochemistry
• Molecular Biology
• Catalysis

Background:

• Existing RNA-cleaving deoxyribozymes (DNAzymes) efficiently cleave purine-containing junctions but struggle with pyrimidine-pyrimidine (Pyr-Pyr) junctions.
• Larger, more complex DNAzyme structures are hypothesized to be necessary for efficient Pyr-Pyr cleavage, but these are difficult to isolate directly from DNA libraries.

Purpose of the Study:

• To derive and characterize novel DNAzymes with enhanced activity for cleaving pyrimidine-pyrimidine junctions, specifically the rC-T junction.
• To investigate the relationship between DNAzyme size, structural complexity, and catalytic efficiency in RNA cleavage.

Main Methods:

• In vitro selection was employed to optimize DNA sequences from existing DNAzymes with weak rC-T junction cleavage activity.
• Chemical mutagenesis was used to create diverse DNA libraries for selection.
• Kinetic analysis (k(obs)) was performed to quantify cleavage rates for various dinucleotide junctions.

Main Results:

• A novel DNAzyme motif, CT10-3.29, approximately 50 nt in size with a complex secondary structure, was isolated with a significant increase in catalytic activity (k(obs) ~0.1 min-1) for the rC-T junction.
• Variants of CT10-3.29 demonstrated further enhanced activity (k(obs) 0.3-1.4 min-1) against the rC-T junction.
• CT10-3.29 exhibited varied cleavage specificities across different dinucleotide junctions, with strong activity for rU-A and rU-T, and altered specificity upon single-point mutation.

Conclusions:

• It is feasible to derive highly efficient DNAzymes for challenging chemical tasks, such as pyrimidine-pyrimidine junction cleavage, through in vitro selection and optimization.
• Efficient RNA-cleaving DNAzymes for difficult substrates require more complex structural solutions than previously known DNAzymes.
• The intricate and plastic nature of DNAzyme catalytic cores allows for modulation of substrate specificity through minor structural alterations.