Fascinating chemistry based on the concept of 'avidity' for sequencing from Element Biosciences described in this recent @biorxivpreprint
It fundamentally differs from sequencing by synthesis (SBS) technology as used in NGS by Illumina, hence it tickled my fancy to write a 🧵:
https://www.biorxiv.org/content/10.1101/2022.11.03.514117v1
Sequencing by synthesis works by sequential incorporation (catalyzed by polymerase) and detection of synthetic nucleotides.
1/n
Practically needs micromolar amounts of enzyme + substrate ie nucleotides conjugated to fluorescent dye (detection) + blocking group (1 base at a time)
Avidity sequencing chemically decouples detection from polymerase extension allowing separate optimizations of each step. That helped reduce working concentration of the new reagents.
The idea of decoupling was also pursued by BGI using antibodies in 2020,
https://www.biorxiv.org/content/10.1101/2020.02.19.953307v1
2/n
Steps involved -
- rolling circle amplification of DNA -> concatemers on flowcell
- imaging 🐙 ie 'avidite': a dye labeled polymer with multiple, identical nucleotide attached -> binds multiple nucleotides across DNA copies
- washing with 🐙 intact -> detection
- remove 🐙 (since held by non-covalent interaction)
- extend 1 base with polymerase + unlabeled, blocked nucleotide
- remove block, repeat
Undiscussed but personally curious about 🐙 design.
4/n
Unrelated (or not), also appending an ode to the thought of a connection my brain made while reading the above with the following figure from https://www.nature.com/articles/s41592-020-0869-x
5/n
Avidity in this context implies the cumulative strength of multiple base pair interactions as opposed to single base pair binding events that SBS relies on.
Stronger interaction means lower substrate concentration needed and longer residence time ➡️ enables imaging without bond formation
Some nice biochemical characterization summarized in the figure below from the paper to crunch kinetic rates from -
3/n