When creating an Oligo workflow in Byos, nucleotide sequences can be added via FASTA file, typed by hand, copy-pasted or built manually using the Oligonucleotide Building blocks sub-tab.
Oligonucleotide building blocks consist of chemical formulas linked to letters or characters. When building a sequence, users can either choose from a list of default components or customize their own.
The list below shows the default building blocks available in Byos:
When customizing a building block, it’s possible to either edit an existing component or create a new one by clicking Add.
The Type column refers to the specific structural component of the building block. The 5' and 3' termini are single-character building blocks with the default formula OH (for terminal hydroxyl groups). Within the sequence, building blocks come in groups of three characters including a nucleobase, ribose, and phosphate linker.
Types:
5' Terminal/ 3' Terminal are single-character building blocks denoted at the 5' terminal by “<” for a hydroxyl group (OH) or “{“ for a phosphate (H2PO4) and as “>” or “}“ for the 3' complement.
Nucleobases include A, C, G, T for deoxyribonucleic acid (DNA) or A, C, G, U in ribonucleic acids (RNA) as well as several methyl, acetyl, and dimethyl variants.
Ribose sugars are defined by including “d” for 2'-deoxy (DNA) or “r” for 2'-hydroxy (RNA) in the sequence as the character preceding the nucleobase. Similar to the nucleobases, several variants such as “m” for 2'-O-methyl and “e” for 2'-O-methoxyethyl are included as building blocks.
Phosphate backbone linkers represented by “.” for a standard phosphate and “*” for a phosphorothioate linker are included as building blocks.
The Oligo workflow allows for full flexibility in defining types of building blocks, any nucleobase can be combined with any sugar and linker to form a sequence. However, a specific order of the building block is required as shown here:
5' terminal RiboseBasePhosphate #1 RiboseBasePhosphate #3………………… 3' terminal
Be aware, by default, if 3’ or 5’ terminal characters are not defined, we assume -OH (5’ “<” and 3’ “>”). If a ribose character is omitted, the default 2'-deoxy (“d”) for deoxyribose is used. Also, if a phosphate linker character is omitted, the default -PO4 (“.”) is assumed. As an example, the DNA sequence shown below can be input as individual bases or be broken down into its building blocks:
CCACGACCAAGTGACAT or <dC.dC.dA.dC.dG.dA.dC.dC.dA.dA.dG.dT.dG.dA.dC.dA.dT>
Nonstandard deoxyribose-based sequences can also be assembled but require additional characters preceding the nucleobase. As an example, the base sequence for Nuisnersen, an antisense oligonucleotide, can be built as follows:
rUrCArCrUrUrUrCArUAArUGrCrUGG
Nusinersen phosphorothioate can be built by replacing the “.” PO4 linker with “*” PO3S for phosphorothioate:
rU*rC*A*rC*rU*rU*rU*rC*A*rU*A*A*rU*G*rC*rU*G*G
And substitution of 2'-O-methoxyethyl for the ribose base:
eU*eC*A*eC*eU*eU*eU*eC*A*eU*A*A*eU*G*eC*eU*G*G
Custom building blocks are added through modification of the existing building blocks or by adding new building blocks. For example, morpholino oligos are non-ionic analogs of DNA molecules with 6-member replacing standard DNA ribose sugars and phosphorodiamidate replacing the phosphate linker. Here, modification of the existing chemical formula for the phosphate group in the building blocks tab would result in the following changes:
PO4 Phosphate linker → C2H6NPO3 Phosphorodiamidate