Measurement

We performed measurements on existing aptamers, including both novel characterizations and attempts to replicate values reported in literature.

Introduction

The protocols for the experiments resulting in each of the measurements reported here is available on the experiments page, to allow for replication or adaptation by other iGEM teams. We also cite relevant sources for our protocols here. For data availability, see the results page.

PFOA Aptamer PFOA_JYP_21

Flurophore-Quencher Affinity Assay

  • Measurement: Binding Affinity (KD) of PFOA_JYP_2 to its target PFOA, more details on experiments page
  • Novelty: Replication of Park, J et al.1
    • Agreement: moderate discrepancy ( ≈ 2 orders of magnitude)
      • Originally reported: 5.5 ⋅ 10−6M
      • Our measurement: 2 ⋅ 10−4M
  • Controls:
    • This was a pilot experiment. It is possible that with more controlled conditions (performing everything within the plate reader, not having to move reagents between labs mid-experiment) we could have gotten closer to the originally reported value. However, the conditions were still generally controlled for.
  • Usefulness:
    • Other iGEM teams and researchers may want to consider double-checking the KD of the aptamer before commiting to it if they are considering using it, because of the moderate discrepancy in binding affinity which, if correct, may be application critical. However, as our experiment was a pilot this is in no way intended to serve as a high-confidence challenge.
    • Our replication does validate that aptamer system does qualitatively bind to PFOA, despite the quantitative discrepancy on binding affinity

Progesterone Aptamer P4G032

A literature search did not reveal any papers mentioning the P4G03 aptamer for progesterone following the paper that originally reported it2, so to our knowledge all results we present that are not replications of Jiménez, G. C. et al. represent novel measurements. The Jiménez, G. C. et al. paper focused most attention to the P4G13 aptamer as they measured it with a greater conformational change when exposed to progesterone, but the P4G03 aptamer had a lower KD value.

Isothermal Titration Calorimetry (ITC) Experiment

  • Measurement: Binding Affinity (KD) of P4G03 sequence to its target progesterone (with no modifications)
  • Novelty: novel measurement method, same value reported in Jiménez, G. C. et al.
    • Although KD values were reported for P4G03 in the original paper2, these values were only calculated for P4G03 using fluoresence, so our use of ITC represents a novel method for calcuclating this value.
    • Agreement: minor discrepancy ( < 1 order of magnitude)
      • Originally reported value (fluoresence): 9.63 ± 3.12 nM
      • Our measurement (ITC): 50 nM
  • Controls:
    • The controls that needed to be run to calculate this KD are buffer only into buffer only showing zero heat changes. Another control of target molecule diluted in buffer into buffer only must be subtracted from experimental run of target molecule diluted in buffer into aptamer diluted in buffer. These were all controls that we fulfilled to correctly calculate the KD. The specific protocol we followed with the concentrations used for our buffers and our experimental conditions is on our experiments page.
  • Usefulness:
    • Other iGEM teams and researchers have a novel validation that P4G03 binds progesterone with binding affinity within the tens-of-nanomolar range
      • this value can be used for calculations and for initial analysis to see if this aptamer is a good fit for the chosen application
      • This may serve as a green light for teams that are looking for a very sensitive aptamer for progesterone
      • the slight discrepancy may be related to differences of measurement methods
    • This measurement does not assess any potential electrochemical modifications of the P4G03 sequence

Circular Dichroism (CD) Experiments

  • Measurement: Qualitative determination, “Does the aptamer display significant conformational change given changing values of target?”
    • qualitative measurement
    • inferred from quantitative data through visualizations
  • Novelty:
    • P4G03 with intended target (progesterone): replication with modifications
      • original experiment reported in Jiménez, G. C. et al. supplementary information used only two concentrations, where no progesterone and 1μM aptamer were compared with 1μM of both progesterone and aptamer.
      • In contrast, our experimental concentrations were higher, with starting concentrations of progesterone at 100μM * (108/280) ≈ 39μM.
      • We tested multiple ratios of progesterone to aptamer.
    • P4G03 with DHT, P4G03 with cholesterol: novel, and no analagous experiments for P4G13 reported in Jiménez, G. C. et al.
      • Cross reactivity experiments in Jiménez, G. C. et al. were focused on making sure P4G13 is specific to progesterone against environmentally relevant hormones. No cross-reactivity experiments were performed for the distinct P4G03 aptamer, which was not selected for further study due to lower conformational change, despite its higher KD.
      • Additionally, no experiments were reported by Jiménez, G. C. et al. investigating the clinically relevant hormones DHT and cholesterol, as the cross-reactivity experiments were focused on environmental applications
  • Agreement:
    • Our CD experiments with P4G03 appear to display greater response conformational change than those in Jiménez, G. C. et al., which may just be due to the increased concentrations and more extreme ratios of aptamer and progesterone that we used.
  • Controls:
    • Aptamer only (to establish the baseline CD signature)
    • Ligand only (progesterone, DHT, cholesterol)
    • Buffer only
    • Ethanol-only solvent control (confirming ethanol concentration did not affect the CD signal)
    • Equal steroid hormone ratios: All ligands tested at identical molar ratios (0.2:1, 5:1, 20:1 in our last experiment) and solvent compositions.
    • The CD spectrometer was zeroed with a clean ethanol/PBS buffer baseline before each scan. Temperature stability was confirmed at 37 °C throughout. No instrumental drift was detected.
  • Usefulness:
    • We were able to confirm some conformational change of P4G03, however the relative magnitude compared with that reported in Jiménez, G. C. et al. is ambiguous due to different test concentrations. Our data may be useful for future analysis to better characterize the conformational change of P4G03 across a wider variety of concentrations.
    • We validated that the P4G03 is specific to progesterone against two important clinical cross-reactants, eliminating potential concerns about this aptamer for future iGEM teams who may desire to use it for health applications.

Alternating Current (AC) Voltammetry Experiments

  • Measurement: the impedimetric response of the electrochemically modified P4G03 aptamer to progesterone in solution
    • essentially changes in a current/voltage graph due to changing solution concentrations of target (progesterone)
  • Novelty: Novel. Similar electrochemical experiments were conducted by Jiménez, G. C. et al. for P4G13 and P4G11 aptamers.
  • Controls:
    • Our data still needs to be improved - we need to have better controls particularly in running the experiment without progesterone compared running it with progesterone over time to determine a change in voltage to eliminate the change of voltage in the instrument to determine if these measurements our useful. That is our top priority at the moment is to improve our controls on this experiment, so that this data can be used to create a baseline of the voltage of an aptamer sensor without any progesterone bound compared to having progesterone bound.
  • Usefulness:
    • we have witnessed some signal from this aptamer, so our qualitative measurement that this aptamer does respond through bio-impedance to progesterone should signal to future iGEM teams that this aptamer may be worth considering for their purposes.

References

  1. Park, J., Yang, K.-A., Choi, Y. & Choe, J. K. Novel ssDNA aptamer-based fluorescence sensor for perfluorooctanoic acid detection in water. Environ. Int. 158, 107000 (2022).

  2. Jiménez, G. C. et al. Aptamer-Based Label-Free Impedimetric Biosensor for Detection of Progesterone. ACS Publications https://pubs.acs.org/doi/pdf/10.1021/ac503639s (2015) doi:10.1021/ac503639s.