A nanophotonic approach to building DNA using enzymatic synthesis

NIH RePORTER · NIH · R01 · $530,297 · view on reporter.nih.gov ↗

Abstract

Abstract Long strand oligonucleotide synthesis continues to be limited by its diminishing returns, with a current maximum length of ~ 250 bases. As a general rule, one of every 100 molecules will fail to couple, meaning that the average synthesis run is said to have a coupling efficiency (CE) of 99%. The formula, CEn, where n is the number of bases added during synthesis, states the longer the strand generated, the more failure strands will be produced. For example, synthesis of a 40 base strand with a 99% CE will generate 68% full-length product (FLP) as opposed to synthesis of a 200 base strand, which will yield 13% FLP with the same CE. While there are other factors that may influence CE (i.e. synthesis parameters and quality of reagents), the main problem is inadequate accessibility of reagent to each of the molecules on the surface of the solid substrate (i.e. polystyrene beads or controlled-pore-glass). The most common case is when beads are packaged inside a column sandwiched between two porous filters; here, stacking of beads causes reduced surface area exposure to synthesis reagents, whereby DNA molecules become unreacted or only partially reacted. Moreover, spent reagents and unwanted byproducts become trapped within the support and carry over into consecutive cycles, further contaminating the synthesis run. To circumvent these limitations, we propose a novel method that allows us to control the actions of an individual bead through dielectrophoresis on a plasmonic surface. Here, reactions are tuned to completely encapsulate each bead with minimal volume reagent droplets for high-precision synthesis. Because each bead is isolated in solution, byproducts cannot become trapped, and each has maximum contact with all synthesis reagents; it is this intimate 1:1 ratio of bead to reagent that will significantly increase the base addition efficiency allowing the production of ultra-long strands of DNA > 1000 bases. Until very recently, far-field optics (i.e. optical tweezers) could not be applied at the nano-scale due to diffraction-limited focused spot size; therefore, researchers began studying effects of plasmonic nanostructures where light waves are concentrated directly onto the bead. In our platform, reagent droplets of precise volume and concentration are formed by pulsed laser cavitation; droplets are then transported along the plasmonic surface to encapsulate individual beads by overcoming surface tension barrier using dielectrophoretic forces generated by an AC electrical field. Thus, this approach of encapsulating a bead into a droplet and pulling it out can be employed for a large range of droplet and bead sizes with the appropriate electrode design. We believe the key to maximizing oligonucleotide purity and yield during synthesis lies in determining the minimal volume/concentration of each reagent necessary to coat the surface of an individual bead. With our proposed platform of synthesis on a plasmonic surface, we have the capabi...

Key facts

NIH application ID
10035169
Project number
1R01GM138716-01
Recipient
STANFORD UNIVERSITY
Principal Investigator
Ronald Wayne Davis
Activity code
R01
Funding institute
NIH
Fiscal year
2020
Award amount
$530,297
Award type
1
Project period
2020-09-23 → 2024-07-31