Modify oligos and RNA sequences

When working with DNA oligos or RNA oligos/sequences, you may need to modify these oligos/sequences at the nucleotide level to enhance activity, stability, or binding affinity. Using Benchling’s monomer library, you can apply these modifications to oligos/sequences and model them on the sequence map. Additionally, you can create custom notation libraries to represent the modifications using your preferred syntax. To learn more about the monomer library and how to configure it, see this help article.

Create new modified oligo

Global create

Note: This option is only available for DNA/RNA oligos

1. Click Global Create and hover over Oligo and click New DNA/RNA oligo from the menu
2. In the pop-up model:
   • Enter a Name
   • Select the Project folder to associate with this entity
   • Select the Study to associate with this entity
   • Select the correct nucleotide type - DNA or RNA
   • Select the Schema to register the entity under
   • Select the Notation you would like to use
     • Note: If you have modified nucleotides enabled, you will have the following options- Natural bases, HELM, and any custom notation configured within the monomer library. To see more about the monomer library and custom notation, see this article
3. Enter your sequence within the Bases field using the selected notation
4. Click Create
   • Note: If you want to add this sequence to your registry, be sure to click the Register button in the upper right corner of the sequence’s metadata data tab

Registry Create

Note: This option is only available for DNA/RNA oligos

1. Click the Registry icon to open the registry application
2. Click the local create button in the upper right corner
3. Hover over the desired schema type (DNA/RNA oligo) and select the entity schema you would like to use
4. In the pop-up modal:
   • Choose your Name and registry ID settings
   • Enter an Entity name
   • Select the Project folder to associate the entity with
   • Fill out the schema fields
   • Select the Notation you would like to use
     • Note: If you have modified nucleotides enabled, you will have the following options- Natural bases, HELM, and any custom notation configured within the monomer library. To see more about the monomer library and custom notation, see this article
5. Enter your sequence within the Bases field using the selected notation
6. Click Register

Registration Table

1. In the Notebook toolbar, click Insert to open the dropdown menu
2. Select Registration table
3. Within the pop-up modal:
   1. Select the desired schema from the Schema dropdown menu
   2. Choose your Name and registry ID settings
   3. Select the Notation you would like to use
      1. Note: If you have modified nucleotides enabled, you will have the following options- Natural bases, HELM, and any custom notation configured within the monomer library. To see more about the monomer library and custom notation, see this article
   4. Click Insert to add the table to your entry

Import modified oligos/ RNA sequences in bulk

Global create

1. Click Global Create and hover over DNA/RNA sequence or Oligo (depending on which you would like to use) and click Import DNA/RNA sequences via spreadsheet or Import DNA/RNA oligos via spreadsheet
2. Within the pop-up modal:
   1. Select the Nucleotide type
      • Note: If using a sequence schema type, it must be an RNA sequence
   2. Click Next
   3. Check that the Import from field has Spreadsheet selected
   4. Upload your spreadsheet and click Next
   5. Map the spreadsheet column headers to the appropriate schema fields. Make sure to correctly identify your sequence field based on the notation used to avoid errors
      • If you select Bases, specify how Benchling should interpret them within the pop-up modal
   6. Click Next
   7. Review/resolve any errors that appear during the validation check to avoid having your import blocked
   8. Click Import

Registry create

1. Click the Registry icon to open the registry application
2. Click the local create button in the upper right corner
3. Click Import entities from the dropdown menu
4. In the pop-up modal:
   • If your tenant has multiple registries, make sure the Registry field has the appropriate one selected
   • Select a Schema
     • Note: If using a sequence schema type, it must be an RNA sequence
   • Select a Project folder to associate these entities with
   • Choose your Name and registry ID settings
   • Click Next
   • Check that the Import from field has Spreadsheet selected
   • Upload your spreadsheet and click Next
   • Map the spreadsheet column headers to the appropriate schema fields. Make sure to correctly identify your sequence field based on the notation used to avoid errors
     • If you select Bases, specify how Benchling should interpret them within the pop-up modal
   • Click Next
   • Review/resolve any errors that appear during the validation check to avoid having your import blocked
   • Click Import

Registration Table

1. In the Notebook toolbar, click Insert to open the dropdown menu
2. Select Registration table
3. In the pop-up modal:
   • Select the desired schema from the Schema dropdown menu
   • Choose your Name and registry ID settings
   • Select the Notation you would like to use
     • Note: If you have modified nucleotides enabled, you will have the following options- Natural bases, HELM, and any custom notation configured within the monomer library. To see more about the monomer library and custom notation, see this article
   • Click Insert to add the table to your entry
4. In the header of the registration table, click the blue plus and select Add from spreadsheet
5. Map the spreadsheet column headers to the appropriate schema fields
6. Click Next
7. Review/resolve any errors that appear during the validation check to avoid having your import blocked
8. Click Import

Note: For more information on how to import entities in bulk, see this article

View modified oligos/sequences

Once you have created a modified oligo within your Benchling tenant, you can use the sequence map of those entities to view the modifications at the nucleotide level. 

In the sequence map, individual nucleotides are represented by capital letters that correspond to the nucleotide’s nitrogenous base. Nucleotides without modifications will have a filled circle around them. Nucleotides with modifications will have the following symbols based on the type of modification:

• Circle Outline: Modified sugar
• Filled circle: Modified base
• Filled diamond: Modified 3’ phosphate
• Diamond dotted outline: Empty 3’ phosphate

Note: The color of each of the above symbols is determined by the color set in the monomer library

To view a nucleotide’s specific modifications within the sequence map, hover over the nucleotide to see a pop-up containing that nucleotide’s specific modifications, displayed as a chemical structure.

Edit modified oligos/sequences

On all modified sequences, there will be a Nucleotides tab. This tab can be used to view each nucleotide's individual modifications at the sugar, base, and 3’ phosphate levels in a list format. 

In this tab, you are able to edit/ apply modifications anywhere within the sequence. When a modification is added or removed, all computed fields on the entity and the sequence map will update accordingly.

Search and filter modified oligos/sequences

Within Benchling’s search tools, you can query for specific modifications to find the sequences you are looking for. Here are filters and the filter options that can be set when searching modified oligos/sequences:

• Nucleotide string
  • Filter condition: includes text, is, or is empty
• Monomer
  • Filter condition: is or is not
  • Select monomer from dropdown list
  • Position: at any position, at position(s), minimum count, maximum count
• DNA HELM String
  • Filter condition: includes text or is

Apply modification patterns

Note: This tool is only available for DNA/RNA oligos

Since modified oligo/sequence based research is rapidly evolving, oligo/sequence modification patterns are often edited or optimized throughout experiments. Benchling has built a modification pattern tool so you can quickly apply a new modification pattern on oligos already within your Benchling tenant. This tool can be used on single oligos or up to 100 oligos at once. To apply a modification pattern:

1. Make sure you have an existing oligo within your Benchling tenant that contains the modification pattern you want to use. This will be used as a template to apply modifications to the selected sequence
2. Within global search or the registry, expand the application to full screen by clicking the directional arrow symbol in the upper right corner of the application pop-out
3. Search for all the oligos you would like to apply your modification pattern to and select them using the blue check boxes on the left hand side
4. Click the More dropdown in the upper right corner
5. Hover over Bulk edit
6. Select Apply modification pattern
7. In the pop-up modal:
   • Select the oligo containing the modification pattern you want to apply to the selected oligo
     • Once you select the oligo, a preview will appear at the bottom of the pop-up modal allowing you to review the modifications
   • Adjust the parameters of the modification pattern:
     • Modification start position: left justify, right justify, or custom start position
     • Toggle on/off Apply base modifications
       • Transfer bases from pattern to target, in addition to sugar and phosphate modifications. Degenerate bases on the pattern are treated as constraints on the target, but will not be applied
     • Toggle on/off Overwrite existing modifications
       • Overwrite any conflicting modifications on the target oligo
   • Resolve any errors that show up within the preview
   • Click Apply

You can also apply modifications to a single oligo by opening the entity, highlighting the area you would like to add the modifications to, right clicking, and selecting Add modifications