To Bead or not to Bead: Struggling to Detect tRNA Binding to Silica Particles towards Wastewater Genome Extraction

ByMichael Klaczko

This post describes my second attempt at developing a protocol for viral genome extraction in large volumes of waste water. Like our first attempt, described here, we looked to improve upon the protocol Direct wastewater RNA extraction via the “Milk of Silica (MoS)” method – A companion method to “Sewage, Salt, Silica and SARS-CoV-2 (4S)”. To inform the design of this experiment, I previously took a look at the mechanism of genome binding to silica beads, shown here. Using what we learned in that deep dive, I developed the experiment shown below. Unlike our first attempt which was more of a “Hail Mary” approach, I take a step back with this experiment in an attempt to develop some ground truths of the system.

Design

The ground truths we looked to establish with this experiment are as follows:

  1. We can bind genomic material to silica beads using our protocol
  2. We can release genomic material from silica beads using our protocol

In addition, we looked to establish an accurate binding capacity for the silica beads we were using to get an idea of the lower and upper bounds of our protocol’s sensitivity. To accomplish these goals, we designed a very simple experiment shown below:

  1. Determine initial tRNA concentration of an aliquot using optical density (OD) measurements
  2. Mix silica beads and tRNA aliquot and allow to interact
  3. Centrifuge mixture to pellet beads
  4. Determine unbound tRNA concentration using OD after interaction with the silica beads
  5. Remove unbound tRNA solution, add water to release bound tRNA
  6. Determine bound tRNA concentration using OD

We decided to use tRNA because it is small (77 base pairs1). We figured this would give us the most accurate binding capacity on a bead since a short RNA strand would not fold over on itself or the bead, blocking potential binding sites for unbound RNS strands. While we could have used qPCR to quantify bound and unbound tRNA, we decided to use our new Nanodrop to take OD measurements since this would be simpler. Unfortunately, both of these design choices ended up making our experimental design very difficult as I had to constantly fight with the limit of detection of the Nanodrop which was made more challenging because of the small mass of tRNA. In order to design an acceptable experiment, I ultimately had to end up using our bead stock concentration and a very large amount of tRNA in order to get noticeable OD changes using the Nanodrop. Important considerations for the design of the experiment are shown below:

  • Assuming a parking space diameter of 3 nm for DNA, a 3 um silica bead can bind 9,424 total tRNA strands
  • Lower limit of detection of the Nanodrop = 1.6 ng/uL
    • 1.6 ng * (1 g / 1E9 ng) = 1.6E-9 g / 25,000 g/mol = 6.4E-14 mol *6.02E23 tRNA/mol = 3.85E10 tRNA
    • Lowest number of tRNA that can be detected by the Nanodrop is 3.85E10 tRNA
  • Upper limit of detection of the Nanodrop= 22,000 ng/uL

Protocol

Beads

  • Bead stock concentration = 3.122E9 beads/mL
  • 100 uL of 3.122E9 beads/mL = 3.122E8 beads
    • 3.122E8 beads * 9,424 tRNA strands = 2.94E12 tRNA
    • 2.94E12 tRNA / 6.02E23 tRNA/mol = 4.89E-12 mol * 25,000 g/mol = 1.22E-7 g * 1E9 ng / 1 g = 122.2 ng

tRNA

  • 0.25 g/25,000 g/mol = 1E-5 mol * 6.02E23 tRNA/mol = 6.02E18 tRNA
  • 6.02E18 tRNA / 2 mL = 3.01E18 tRNA/mL in tRNA stock
  • 1 uL of 3.01E18 tRNA/mL into 999 uL of nuclease free water = 3.01E15 tRNA/mL
  • 3.01E15 tRNA/mL * V1 = 2E14 tRNA/mL * 1 mL
    • V1 = 0.066 mL = 66.45 uL of 3.01E15 tRNA/mL into 933.55 uL of nuclease free water
    • 2E14 tRNA/mL Is 2E11 tRNA/uL
    • 100 uL of 2E14 tRNA/mL is 2E13 tRNA
    • 2E13 tRNA/6.02E23 tRNA/mol = 3.32E-11 mol * 25,000 g/mol = 8.31E-7 g
    • 8.31E-7 g *1E9 ng/g = 830.56 ng/100 uL = 8.31 ng/uL
  • If all works out the initial solution will read 8.31 ng/uL

Incubation

  • Add 23.75 mg of NaCl to 100 uL of tRNA solution
    • 100 uL / 40,000 uL = 0.0025 = 0.25%
    • 0.25% * 9.5 g = 0.02375g = 23.75 mg of NaCl
  • Add 1 uL of TE buffer to the tRNA solution
    • 100 uL / 40,000 uL = 0.0025 = 0.25%
    • 0.25% * 400 uL = 1 uL of TE buffer
  • Agitate sample until all NaCl dissolves in tRNA
  • Vortex or shake sample for 30 seconds to promote lysis
  • Add 100 uL of 70% ethanol to the sample
  • Agitate sample to mix ethanol and sample
  • Add beads to the tRNA solution, 100 uL of bead solution
  • Invert tube with lysate & silica 10 times to mix. Incubate mixture at room temperature for 10 minutes.

Unbound tRNA Extraction

  • Centrifuge tubes containing silica & bound RNA at 4000 x g, 4°C, 5 minutes
    • The silica will form a firm pellet at the bottom of the tube.
  • Remove the tubes from the centrifuge and decant the supernatant.
  • Take OD of the supernatant to get tRNA concentration
    • Supernatant volume should be ~301 uL
    • If max hypothetical extraction, should be 2.35 ng/uL (830.56 ng – 122.2 ng = 708.36 ng / 301 uL)
    • If no extraction occurs, should be 2.76 ng/uL (830.56 ng/301 uL)

Bound tRNA Extraction

  • Add 50 uL of 37 °C pre-warmed pure water to pellet and vortex to resuspend
    • Allow to incubate for 10 minutes
  • Centrifuge tubes containing silica & eluted RNA 4000 x g, 37°C, 5 minutes.
    • The silica will form a firm pellet at the bottom of the tube and the RNA will be present in the aqueous phase.
    • Pipette or decant the aqueous supernatant into a sterile conical tube
  • Take OD of the filtrate to get tRNA concentration
    • If max hypothetical extraction has occurred and all tRNA is released, OD should read 2.44 ng/uL (122.2 ng / 50 uL)

Results

The initial tRNA solution measured an average of 6.4 ng/uL, or 640 ng total. This was 1.91 ng/uL lower than it was supposed to be. Measuring the unbound tRNA solution gave an average of 2.6 ng/uL. Using our stock concentration of 6.4 ng/uL, max hypothetical extraction should give a concentration of 1.73 ng/uL (640 ng – 122.2 ng = 521.8 ng / 301 uL). No extraction would give a concentration of 2.06 ng/uL (640 ng/301 uL). Since we measured higher than this, our data suggests we bound no tRNA. This was supported after I was unable to measure a concentration for the bound tRNA.

Conclusions and Future Work

Failure to hit any of our goals in this experiment left us circling the wagons. Moving forward we plan to attempt this approach once more using a higher bead concentration. This would enable us to use more tRNA, allowing us to use a more easily detectable concentration by the Nanodrop. This would also help provide a more noticeable change in initial tRNA concentration if binding is in fact occurring, making any attempt at binding by the silica beads more easily recognizable. The success of this approach relies on the assumption that binding is in fact occurring, we are just missing it because we are working so close to the limit of detection of the Nanodrop. If these problems persist after moving away from the limit of detection of the Nanodrop, we will most likely look to make changes to our binding protocol next.

References

  1. Holley, R. W. et al. (1965) Structure of a Ribonucleic Acid. Science (American Association for the Advancement of Science). [Online] 147 (3664), 1462–1465.

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