Introduction to Modified Oligonucleotides

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Oligonucleotides, or oligos, are short strands of DNA or RNA that are used for a variety of laboratory and clinical applications including molecular biology research, next generation sequencing, and the development of novel therapeutics. In recent years, RNA oligonucleotides containing synthetic modifications have become an area of intense research in pharmaceutical development due to the ability of these molecules to manipulate gene expression to potentially treat disease.

An oligonucleotide is a nucleotide that contains one or more synthetic modifications to its phosphate, ribose sugar, or nitrogenous base. The purpose of these modifications are to confer enhanced activity, stability, or binding affinity to the oligonucleotide sequence.

There are many different types of chemical modifications that are commonly made in an oligonucleotide sequence. Some examples include Phosphorothioate (modification to the 3’ Phosphate), 2’-Fluoro or 2’-O-methyl (modifications to the ribose sugar), and Pseudouridine or 5-methylcytosine (modifications to the nitrogenous base).

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HELM: A Hierarchical Editing Language for Macromolecules

Modified oligonucleotides may contain a large number of synthetic modifications resulting in a complex chemical structure that may be difficult to visualize or interpret using standard chemical notation or the sequence alone. HELM (Hierarchical Editing Language for Macromolecules) is a scientific language that was developed to solve this issue, by standardizing the representation, registration, and visualization of these complex chemical structures (including oligonucleotides) for the wider scientific community. HELM notation is applicable to DNA, RNA, AA, and chemical compounds to model siRNA, ADCs, modified peptides, and more.


Representative linear oligonucleotide visualized by Monomer Graph View (A), with its corresponding HELM notation (B). Figure from Zhang et al., J. Chem. Inf. Model 2012.

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HELM defines a hierarchy to describe any AA, RNA, or DNA polymer with a method to connect them together.

HELM has become a valuable method that enables scientists to represent the chemical constituents of a modified nucleotide sequence. However, the adoption of HELM across the scientific community is not complete, and there is no standard file format or syntax that is widely agreed upon by vendors that synthesize oligonucleotides. Further, currently available visualization tools are not very intuitive for scientists working at the bench.

Modified Oligonucleotides in Benchling

Benchling has developed a set of tools that reinvent how modified oligonucleotides are visualized and represented in-silico, enabling scientists to quickly and easily keep track of their oligo modifications within the Benchling platform. Here, we’ve developed a new “sequence-centric” method for visualizing phosphate, sugar, and base modifications, with the ability for scientists to easily introduce new modifications to their sequence with Benchling’s Monomer Library.

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The Monomer Library

Benchling’s Monomer Library houses all of the modifications that can applied to the individual monomers of a nucleotide sequence: these include base, sugar, and phosphate modifications. The Monomer Library is accessible via Benchling’s Registry Settings, where users can see which modifications are available to apply to an oligo sequence. Non-standard or custom chemical modifications can also be created in the Monomer Library. Contact Benchling Support for more information.

Working With Modified Oligonucleotides in Benchling

For instructions on creating, editing, and visualizing oligo modifications within Benchling, please view the help articles included in the Modified Oligonucleotides Collection.

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