| Criteria | RNase A | RNase H | Remarks |
| Full Name | Ribonuclease A | Ribonuclease H | Both are RNases but differ in substrate specificity and biological function |
| Substrate Specificity | Cleaves single-stranded RNA at the 3′ end of pyrimidine residues | Specifically degrades RNA in RNA:DNA hybrids | RNase A acts on free ssRNA, RNase H targets RNA hybridized with DNA |
| Source | Commonly derived from bovine pancreas | Found in both prokaryotic and eukaryotic cells | RNase H exists naturally in most organisms; RNase A is widely used as a lab reagent |
| Cleavage Mechanism | Endonuclease activity on ssRNA | Endonuclease activity only on RNA strand of RNA:DNA duplex | RNase H cannot act on single-stranded RNA or double-stranded RNA |
| Structural Characteristics | Small, stable, and robust protein | Composed of multiple isoforms (H1 and H2 in eukaryotes) | RNase H has multiple subtypes with distinct functions |
| Biological Role | Degradation of extracellular RNA; part of RNA turnover and defense | Essential in replication and repair, particularly in removing RNA primers | RNase H is important in retroviral replication and Okazaki fragment processing |
| Biotechnological Use | Removal of RNA from samples; mRNA cleanup | Removal of RNA after reverse transcription | RNase H is routinely used in cDNA synthesis protocols |
| Activity Conditions | Functions optimally at neutral pH and in presence of divalent cations | Requires Mg²⁺ or Mn²⁺ for activity | Metal ions are critical for both enzymes, but RNase H is more metal-dependent |
| Inhibition Sensitivity | Sensitive to RNase inhibitors (e.g., RNasin) | Partially resistant to RNase A inhibitors | Inhibitor compatibility must be considered in experimental setups |
| Application in Molecular Biology | General RNA degradation, purification processes | Used in RNA-Seq prep, cDNA synthesis, and antisense studies | Applications are substrate-specific; choosing the right RNase is critical for experiments |
