Modification Patterns

Benchling’s Modified Nucleotide functionality to model oligonucleotide therapies such as siRNA or ASOs based therapies can be used to rapidly create and design modified entities and continue to iterate and innovate on current modification patterns by using the “Modification Pattern” from an existing Oligo and applying that pattern to a batch of natural target oligos.


The purpose of this tool is to allow users to easily apply modifications from a pre-existing oligo to 1 or more target oligos.

When bulk applying a Modification Pattern, we currently impose a max batch size of 100 target oligos at a time.

A message will appear if the modification pattern is applied to a batch size > 100


The tool can be used to apply a modification pattern onto

• a single target Oligo - from the Nucleotide’s tab of the target Oligo itself
• 
• a batch of selected targeted Oligos - from the expanded listings (Project, Registry, or Search) page
• 

Choosing a pattern

After selecting the option to “Apply Modification Pattern”, users are taken to a modal where they must first Choose a Pattern off of a pre-existing oligo.

The Choose Pattern screen consists of 2 main components

• The top half is a search listing component that lets users search across any oligo of the same nucleotide type as the target(s) selected.
  • The search listing components lets users search by name, as well as filter by Schema, Folder, or any of the options you have access to from expanded listings.
• The bottom half consists of a Preview Pattern component that displays the modification pattern, along with a monomer visual legend, of any Oligo you have selected.

Once you have selected an existing oligo to use, you can select Next to progress to the next screen.

Applying Modification Patterns

On the next step of the modal, users have an opportunity to change some parameters that will affect how the modification pattern gets applied to their target oligo(s).

Users have the option of toggling:

• Where the modification pattern snaps to, relative to the target oligo via the Justify setting
• Whether or not to Apply base modifications
• Whether or not to Overwrite existing modifications

Detailed explanation of parameters to apply pattern to targets

Left/Right Justify

• This setting determines the starting position on the target oligo where the modification pattern will be applied.
• Left: The pattern will snap to the left of the target so that the first base (3’ end) of the modification pattern lines up with the first base (3’ end) of the target(s)
• Right: The pattern will snap to the right of the target so that the last base (5’ end) of the modification pattern lines up with the last base (5’ end) of the target(s)
• Custom: The pattern will be applied starting at the user-defined nucleotide position on the target oligo - the first base (3’ end) of the modification pattern will be lined up with the custom start position provided by the user.

Applying Base Modifications

• When creating/saving an oligo that is intended to be used as a modification pattern, users can utilize degenerate bases to define these compatibility constraints for targets or simply to denote positions where they want to leave existing bases.

Modification Pattern from Template Oligo	Target Oligo bases	Apply Base Modifications	Expected result	Result Explanation
	AAAAA	Do Not Apply		The "Apply Base Modifications" setting is not applied, and thus only the ribose and phosphate modifications are applied from the Modification Pattern Template Oligo.
	AAAAA	Apply		All base modifications are applied to the target - changing A's to CCGGC, as specified in the Modification Pattern Template Oligo.
	AAAAA	Apply		Degenerate bases on the Modification Pattern Template Oligo are used for compatibility validation only - because all degenerate bases in the Pattern are N's, the validation passes against our Target's bases and the pattern is applied.
	AAAAA	Apply		The degenerate base B stands for "Any base EXCEPT Adenine". Attempts to apply the base modifications will show an error message, and the user will not be allowed to apply this modification pattern on a target of AAAAA because it violates the compatibility validation check.

As you adjust the parameter settings, the bottom Target Previews Table refreshes to give you a preview of how the target oligo(s) will be modified.

• The portion highlighted in yellow denotes the region on the target oligo(s) where the modification pattern is being applied to
• Any bases shown with a purple underline denote the bases that will actually be changed by applying the modification pattern
• If there are any expected errors, the messages are surfaced in warning icon tooltips in the target previews table.

When you’re satisfied with the configurations, you can click apply to start the editing operation.

• This may take up to a few minutes if you’re working with many targets in bulk.
• Upon completion, the modal will automatically close and return to the entity tab for the single target flow, or Benchling will display the results table in the bulk targets flow so that you can review the results.

Bulk Applying a modification pattern to multiple targets

To apply a pattern to multiple oligos, you should select the target oligos in an expanded listing, and access the modification pattern menu option under More→Bulk edit→Apply modification pattern. Note that the option will only be available if the following criteria are met:

• All selected entities are oligos of the same nucleotide type
• There are no more than 100 targets selected

The overall user flow of Bulk Applying modifications will mirror the single target flow:

Applying a Modification Pattern workflow to a single target oligo with some small differences:

When attempting to Bulk Apply a modification pattern:

• The bulk operation will attempt complete even if individual targets produce errors.
• The bulk operation will only be blocked from submission if we expect all targets to fail
• Oligos that were successfully edited with the applied pattern will have a green checkmark next to them.
• A summary banner will be displayed to show how many of the targets were successfully edited and how many failed.

Overwriting existing modifications

• If this setting is left unchecked (default)
  • These conflicts are treated at the monomer level, so an existing sugar modification on the target would not conflict with a phosphate modification on the applied pattern.
  • Eg. position 5 on the target has 2omethylribose, but position 5 on the modification pattern reads 2fluororibose. If the setting is left unchecked, then we will NOT overwrite to 2fluororibose.
• Any pre-existing modified monomer on the target oligo that conflicts with what is specified at that position on the modification pattern will be left as is
• If the setting for “Overwrite existing modifications” is checked, then all pre-existing Phosphate and Ribose modifications will be ignored on the target and transfer the exact Ribose and Phosphate modifications specified on modification pattern.
• Note that this means that a position where the target has an existing modification but the pattern does not, the resulting position will not have any modifications.