Introduction
Our contribution for future iGEM teams is to quantify the sensitivity, kinetics and stability of IDT RNaseAlert™ probes, a commercially available probe that can be used with Cas13a diagnostic devices, to provide other teams working with either Cas13a or RNase based experiments to reduce cost and minimize the use of specialist equipment. To quantify these traits, our team performed a series of experiments measuring:
1) The effect of varying concentrations of RNase A has on levels of fluorescence intensity and speed kinetics (Vmax and Kmax) measured when RNAseAlert™ is cleaved.
2) The relationship between IDT RNaseAlert™ probe concentration and fluorescence. This will allow for cost-effective device design my minimizing usage of expensive probe components.
3) The effect of various environmental conditions on the level of RNase contamination, allowing teams to determine whether their test will be robust in field environments, and whether specialist equipment, including RNAse free sterile hoods are required.
RNAse A Concentration Experiments
Method
A stock solution of RNase A (730 μM) was prepared and a 10x serial dilution was performed in QIAGEN nuclease-free water. All experiments were performed in 200μl volumes in a 96 well plate (containing 148 μl of tris-HCl 250 mM elution buffer and 50 μl of IDT RNaseAlert™ probe), with fluorescence read using a Tecan Infinite m200 Pro (using the protocol available in (LINK to measurements?). A measurement was taken at 0 minutes (488 nm excitation, 519 nm emission), and then every 5 minutes for 20 minutes.
Results
[Figure 1]A graph showing the change in fluorescein concentration equivalent (nM) of IDT RNaseAlert™ probe over time for a series of 10x RNase A dilutions. The negative control (blank) consists of QIAGEN nuclease free water (200 µL). Error bars show 5 biological repeats.
[Figure 2] A graph showing the changing Vmax (nM min-1) of different RNase A concentrations (nM).
As shown [Figure 1], increasing the concentration of RNase A enzyme increases both the fluorescence intensity and rate of reaction [Figure 2]. The high R-squared value [Figure 2] shows that our data closely follows our Michaelis-Menten modelling, suggesting it can act as a positive control to show that the RNase component of our system (the Cas13a enzyme) works successfully. This can also be used to quantify the kinetics of a Cas13a reaction compared to a known standard. As part of our contribution, future teams may find this modelling useful in assessing the efficacy of their RNase, or the kinetics of Cas13a parts.
Although the highest concentrations of RNase produce the strongest fluorescence, future teams may decide to use this data to optimize their RNase concentration to balance the required level of fluorescence, rate of reaction and resource use based on their system’s individual needs. Furthermore, these results highlight the sensitivity of IDT RNaseAlert™ probes to minimal levels of RNase contamination, prompting further investigation into the experimental effect of environmental RNase contamination (see Section 3).
Fluorescent Probe Concentration Experiment
Method
IDT RNaseAlert™ probes were prepared with QIAGEN nuclease-free water to 250, 125, 65, 30, 15, 10 and 5 nM concentrations. All experiments were performed in a 96 well plate (containing 2 µL of RNase A and made up to 200 µL with tris-HCl 250 mM elution buffer) with fluorescence read using a Tecan Infinite m200 Pro using the protocol available in our Measurement page. The negative control (blank) contains 200 μl of nuclease-free water. A measurement was taken at 0 minutes (488 nm excitation, 519 nm emission), and then every 5 minutes for 20 minutes.
[Table 1] A Table showing the concentration of IDT RNaseAlert™ probe and the volume of QIAGEN nuclease-free water, tris-HCl 250 mM elution buffer and RNase A in each well
Results
[Figure 3] A graph showing the change in fluorescein concentration equivalent (nM) of decreasing concentrations of IDT RNaseAlert™ probes exposed to 2 µL of RNase A over time (minutes). Dotted lines show the data produced, whereas solid lines show the Michaelis-Menten model.
As shown [Figure 3], both the fluorescence intensity and rate of reaction are proportional to the concentration of IDT RNaseAlert™ probe used. All concentrations of probe except for 5nM produce a significant R-squared value, showing close adherence to our Michaelis-Menten modelling. Although the highest concentrations of IDT RNaseAlert™ produce the strongest fluorescence at the quickest rate, future teams may decide to use this data to optimize their probe usage to balance the required level of fluorescence, rate of reaction and resource use based on their system’s individual needs. This would allow teams to minimize extraneous costs. For example, a system with a high level of Cas13a may be able to utilise lower amounts of probe, without sacrificing reaction rate or maximum fluoresnce. Systems with a lower amount of Cas13a available may require higher probe concentrations. Furthermore, this experiment enabled us to select a probe concentration that not only produced a discernible fluorescence signal amidst potential environmental RNase contamination but also provided a reliable timeframe for detecting that signal. Given that stakeholders have consistently highlighted the importance of producing rapid tests that avoid producing devastating false positives, this experiment allowed us to develop a quantitative method of selecting the probe concentration that best meets stakeholder needs.
Environmental RNase Contamination Experiments
Method
QIAGEN nuclease free water, MilliQ filtered water, a tris-HCl equilibration buffer (non-sterile) and a tris-HCl 250Mm imidazol elution buffer (non-sterile) were utilized due to their common use across labs, although teams looking to measure contamination in project-specific buffers could repeat the experiment using the protocol available on our Experiments page. 250 nM of IDT RNAseAlert™ probe was added to each well, before a 0 minute measurement was taken (488 nm excitation, 519 nm emission). This establishes the initial level of contamination present in the buffer. One uncovered plate was placed on the lab bench, and the other within a laminar flow hood used solely for RNA work. Further measurements were taken every 10 minutes for two hours, and then another reading was taken after the plates had remained out overnight to measure the presence of RNase in the environment.
Results
[Figure 4] A graph showing the change in fluorescein concentration equivalent (nM) of IDT RNaseAlert™ probes suspended in nuclease-free water, MilliQ water, elution buffer and equilibration buffer left in both the laminar flow hood and on the bench over time (minutes)
As shown [Figure 4] both nuclease-free water and MilliQ (in both the laminar flow hood and on the lab bench) show low levels of contamination, with a negligible increase over 900 minutes. However, both non-sterile elution buffer and equilibration buffer show significant levels of environmental RNase contamination in both the laminal flow hood and lab bench environments. Elution buffer in both environments shows an increase in contamination over time- notably, despite significant differences in starting fluorescein concentration equivalents, both points intersect at 900 minutes. Given the low effect of environmental exposure on nuclease-free water and MilliQ, it is possible both samples of elution buffer contain a similar level of endogenous RNase but an unknown external factor led to an inhibition of the RNase in the sample left on the bench environment. This would require repeat experiments to explore, but due to budget and time constraints this was not possible in our laboratory. Ideally, this experiment would have been conducted in a third environment (outside) to best mimic the environment our test would be conducted in, but due to ethical consideration and stringent adherence to iGEM guidelines, this was not possible.
Overall, the results from this experiment suggests that teams using nuclease-free water or MilliQ do not need to perform their RNase sensitive experiments in specialized environments or take non-standard measures to prevent contamination. This could significantly reduce costs for teams who may not have access to laminar flow hoods. When using buffers that are not nuclease-free, teams should consider using the protocol provided to establish the level of RNase present in specialized buffers to determine whether it is significantly effecting their results.
