454.Bio (Jonathan Rothberg’s new sequencing startup) has been pretty stealthy regarding how the technology actually works. But some patents are now starting to appear!
The approach proposed appears to have some similarities to Reticula, and I thought it would be fun to briefly review.
454.Bio
Here’s the abstract from the 454 patent:
“A method for nucleic acid sequencing, comprising: contacting a substrate polynucleotide immobilized to a substrate with a protected nucleotide and a sequencing primer in a presence of a polymerase such that the polymerase incorporates the protected nucleotide into the sequencing primer, wherein the protected nucleotide comprises a detectable moiety and a photocleavable terminating moiety; using evanescent wave imaging to identify the protected nucleotide incorporated into the sequencing primer; and using evanescent wave imaging to cleave the photocleavable terminating moiety of the protected nucleotide.”
What does this mean in plain English? Well they seem to be suggesting using nucleotides with photocleavable terminators, like lightning terminators1.
From everything I’ve seen 454.bio appears to be trying to build a very cheap/simple sequencing platform, with a $99 price point:
Reducing device and consumable complexity would be critical. An evanescent field and photocleavable terminators would allow you to deactivate terminators/labels near the surface using light.
You don’t need to flow in any reagents to perform a sequencing cycle. And no fluidics makes for a much simpler instrument and consumable.
Critically you must only expose those strands under synthesis near the surface to the cleavage wavelength (here UV). Otherwise you would end up removing terminators from all the nucleotides in the bulk of the solution.
An evanescent field allows you to do this, by confining the light near the surface (where the sequencing is occurring).
Reticula
So, what’s this got to do with Reticula? Well the basic Reticula approach was also to deactivate labels near the surface using an evanescent field.
The approach I’ve previously described was to photobleach nucleotides under a TIRF field as discussed in the Reticula patent:
“Some embodiments, an evanescent light field can be created by total internal reflection of a light beam at an angle larger than the critical angle. In some embodiments, an evanescent field of a laser can be provided through TIRF illumination. In some embodiments, the bleaching laser field can be confined near the surface of the substrate (e.g., within about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, or about 200 nm of the surface)”
Going single molecule enabled Reticula’s use of photo-bleaching but photo-cleavage was always on the cards2 :
“The method of any of claims 1-48, wherein the deactivating in step b) comprises photobleaching a detectable label, photolysis of the detectable label, photocleavage of a photocleavable linker linking the detectable label, temperature-based detectable label deactivation, pH-based detectable label deactivation, or any combination thereof.”3
Summary
The key thing is we both seem to have been looking at deactivating/removing labels near the surface using an evanescent field. This is what lets you perform continuous sequencing without fluidics and in a major benefit of both approaches!
I think I had a decent head start on 454.bio. I filed the first provisionals in 2021. 454.bio filings seem to come from April 2022…
Nice to know Reticula was ahead of the curve!
But kind of a shame that the Reticula patents may end up expiring this December!
And look, the final author of the lightning terminator paper is a 454.bio co-founder!
I just likely wouldn’t have had access to photo-cleavable labels/terminators for the proof-of-concept/seed stage.
“In any of the embodiments herein, the deactivating step may comprise photobleaching a detectable label, photolysis of the detectable label, photocleavage of a photocleavable linker linking the detectable label, temperature-based detectable label deactivation, pH-based detectable label deactivation, or any combination thereof.”
“In some embodiments, the bleaching laser field can be an evanescent light field. In some embodiments, an evanescent light field can be created by total internal reflection of a light beam at an angle larger than the critical angle. In some embodiments, an evanescent field of a laser can be provided through TIRF illumination. In some embodiments, the bleaching laser field can be confined near the surface of the substrate (e.g., within about 50 nm, about 75 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, or about 200 nm of the surface) such that energy is spatially concentrated in the vicinity of the substrate.”
“any of the embodiments herein, the method can further comprise controlling the distance of the deactivation from a surface of the substrate. In any of the embodiments herein, the method can comprise using an evanescent light, an electric field, heat, a change in pH or any combination thereof that is local to the surface of the substrate.”
Thanks for the writeup Nava. Some thoughts:
1) I remember Jonathan Rothberg tweeting (or should we say X'ing?) for LLM experts to apply to 454 Bio. The impression I came away with was that it was more of a QuantumSi type of approach applied to DNA, rather than Lasergen. On that note, why did Agilent kill Lasergen anyway? In their papers Lasergen mention UV damage, could that be a factor?
2) If you have a 200nm depth TIRF and a 1um square pixel, you have a volume of ~0.2fL. If your nucleotides are at 1uM, then you'll have 2*10^-22 moles or about 120 nucleotides in the illuminated volume. So there will be a good deal of background. I guess that's where PacBio's ZMWs come in handy.
3) What kind of throughput would be feasible with this method? PacBio, which is somewhat similar, also has a throughput problem...