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Cell Biology JoVE (Open Access) Citable · DOI

DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

DOI: 10.3791/59700-v
What you'll learn
  • ✓ Design DNAzymes to target specific RNA methylation sites
  • ✓ Isolate and prepare yeast RNA for methylation analysis
  • ✓ Perform DNAzyme-mediated cleavage and analyze methylation patterns
  • ✓ Evaluate snoRNA activity through site-specific 2'-O-methylation detection
Protocol

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2’-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Difficulty
advanced
Total time
~4–6 hours (yeast culture + RNA isolation + DNAzyme digestion + analysis)
Model organism
Saccharomyces cerevisiae
Biosafety
BSL-1

Steps

1
Design DNAzymes for target RNA sequences

Select and design 10-23 and 8-17 DNAzymes complementary to specific rRNA 2'-O-methylation sites. Configure Watson–Crick base pairing and catalytic core sequences for optimal cleavage efficiency.

▶ 00:29
2
Grow S. cerevisiae and isolate total RNA

Culture S. cerevisiae to appropriate density and extract total RNA using standard phenol–chloroform or column-based methods. Quantify and assess RNA integrity.

▶ 01:46
3
Incubate RNA with active DNAzymes

Mix purified rRNA with pre-annealed DNAzyme–substrate complexes under optimized pH, salt, and temperature conditions to enable site-dependent RNA cleavage at 2'-O-methylated positions.

▶ 02:22
4
Purify cleaved RNA and resolve by electrophoresis

Remove DNAzymes and proteins from reaction mixtures via phenol extraction or spin columns. Load samples on denaturing polyacrylamide or agarose gels to separate and visualize cleavage products.

▶ 05:07
5
Interpret methylation patterns and snoRNA activity

Compare cleavage band patterns between 10-23 and 8-17 DNAzymes to map site-specific 2'-O-methylation and infer snoRNA-guided modification efficiency at target rRNA positions.

▶ 07:27
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