# A nanophotonic approach to building DNA using enzymatic synthesis

> **NIH NIH R01** · STANFORD UNIVERSITY · 2022 · $435,297

## 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:** 10460609
- **Project number:** 5R01GM138716-03
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Ronald Wayne Davis
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $435,297
- **Award type:** 5
- **Project period:** 2020-09-23 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10460609

## Citation

> US National Institutes of Health, RePORTER application 10460609, A nanophotonic approach to building DNA using enzymatic synthesis (5R01GM138716-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10460609. Licensed CC0.

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