The table below includes common oligonucleotide digest enzyme expected cleavage sites and recommended Byos Digested Oligonucleotide Workflow settings assuming primary cleavage sites.
Enzyme | Primary*** Cleavage Rules | Byos Workflow Digest Settings | Byos Workflow Terminus Modification Settings |
| RNase T1 | G|N 5' OH and 3' O4PH2 | Cleavage Site(s): G Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed Phospho 3' / +79.966331 @ CTerm | fixed |
| RNase A | C|N and U|N 5' OH and 3' O4PH2 | Cleavage Site(s): CU Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed Phospho 3' / +79.966331 @ CTerm | fixed |
| RNase U2 | A|N 5' OH and 3' O4PH2 | Cleavage Site(s): A Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed Phospho 3' / +79.966331 @ CTerm | fixed |
| hRNase 4 | U|G and U|A 5' OH and 3' O4PH2 | Cleavage Site(s): (U)([AG]) Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed Phospho 3' / +79.966331 @ CTerm | fixed |
| hRNase 4 + T4 PNK | U|G and U|A 5' OH and 3' OH | Cleavage Site(s): (U)([AG]) Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed % Phospho 3' / +79.966331 @ CTerm | fixed |
| RapiZyme MC1 (pH=8) | A/U/C|U 5' OH and 3' O4PH2 | Cleavage Site(s): ([AUC])(U) Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed Phospho 3' / +79.966331 @ CTerm | fixed |
| RapiZyme Cusativin (pH=9) | C|A/U/G 5' OH and 3' O4PH2 | Cleavage Site(s): (C)([AUG]) Cleavage Side: C-terminal | Hydroxy 5'/ -79.966331 @ NTerm | fixed Phospho 3' / +79.966331 @ CTerm | fixed |
| MazF | A|CA 5' O4PH2 3' OH | Cleavage Site(s): (A)(CA) Cleavage Side: C-terminal | % Hydroxy 5'/ -79.966331 @ NTerm | fixed % Phospho 3' / +79.966331 @ CTerm | fixed |
*** Enzyme rules listed are the primary expected cleavage sites. Depending on the enzyme and the sample prep, other digest sites could be possible.
The Byos Workflow Digest and Terminus Modification Settings are entered in the Byos Digest Oligonucleotide Workflow as shown below.
Quick Tips:
Setting the Digestion Specificity to Fully Specific (fast) requires both ends of digest products to follow the set cleavage rules. With Semi specific (slow) at least one cleavage end must follow the set digest rules. Semi specific can allow detection of digest products resulting from offsite cleavages beyond the primary expected cleavage sites, which can be useful for enzymes digestion with off-target cleavages. Non specific (slowest) allows cleavage at any site regardless of the input Cleavage Site(s).
Variable Peptide C-term and Peptide N-term modifications can be added, when >1 3' or 5' terminus is expected. In the figure above, by including the Cyclic Phosphate / +61.955766 | Peptide C-term rare1 modification, the search will allow detection of digest products with 3' hydroxy and 3' cyclic phosphate.