A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Enzymes
2.3. Recording of Spectra
2.4. Investigating the pH Dependence of Spectra
2.5. Enzymatic Reactions
2.6. Enzyme Reaction Sampling
2.7. Spectral Unmixing
- = representation of the molar fractions of compounds for sample k in a vector form (in this study: )
- = experimentally determined spectrum for sample ,
- = number of compounds (in this study: ),
- = molar fraction of compound , and
- = spectra of pure compound , normalized to the corresponding isosbestic point of the mixture (see below),
2.8. Normalization of Spectra to Accommodate Concentration Differences
- = the normalized spectrum of compound ,
- = raw spectrum of compound ,
- = raw blank spectrum of NaOH solution,
- = the absorption of the blank-corrected spectrum at wavelength .
2.9. Statistical Analyses
- = residual sum of squares of all data points;
- = total sum of squares of all data points;
- = predicted molar fraction of the data point of the data set (i.e., all data points for one compounds, considering both replicates), in percent,
- = average of all actual molar fraction values of the data set (equal to 50%), in percent,
- = actual molar fraction value for the data point in percent,
- = total number of data points in the data set,
2.10. HPLC Analysis of Samples
3. Results
3.1. Nucleoside–Nucleobase Pairs Show Discriminable UV/Vis Spectra under Alkaline Conditions
3.2. Spectral Unmixing Can Resolve the Composition of Nucleoside–Nucleobase Mixtures
3.3. The Accuracy of Spectral Unmixing Holds up Excellently across All Possible Mixture Compositions
3.4. Spectral Unmixing Empowers Practical Applications in Enzymatic Conversion Research
3.5. The Spectral Unmixing Algorithm Can be Fine-Tuned to Specific Areas of Interest
4. Discussion
4.1. The Prerequisites of Spectral Unmixing are Met by All Nucleoside–Nucleobase Pairs
4.2. Previous Assays in the Field
4.3. Improvements and Adaptations of the Assay
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Compound | Nucleoside [nm] | Base [nm] | Isosbestic Point for Base Cleavage [nm] | Upper Limit of Range for Fitting [nm] | |
---|---|---|---|---|---|
Pyrimidines | Uridine | 262 | 281 | 271 | 310 |
2’-Deoxyuridine | 262 | 272 | 310 | ||
5-Methyluridine + | 267 | 290 | 277 | 320 | |
2’-Deoxythymidine $ | 266 | 278 | 320 | ||
5-Fluorouridine * | 269 | 281 | 282 | 325 | |
2’-Deoxy-5-fluorouridine * | 268 | 280 | 325 | ||
5-Bromouridine | 276 | 290 | 283 | 330 | |
2’-Deoxy-5-bromouridine | 275 | 282 | 330 | ||
5-Iodouridine * | 281 | 291 | 283 | 340 | |
2’-Deoxy-5-Iodouridine * | 279 | 282 | 340 | ||
5-Ethynyluridine * | 285 | 298 | 262, 288 | 340 | |
2’-Deoxy-5-ethynyluridine * | 284 | 262, 288 | 340 | ||
Cytidine # | 271 | 281 | 271 | 310 | |
2’-Deoxycytidine # | 271 | 271 | 310 | ||
Purines | Adenosine | 259 | 268 | 267 | 310 |
2’-Deoxyadenosine | 259 | 267 | 310 | ||
Guanosine | 264 | 273 | 279 | 310 | |
2’-Deoxyguanosine | 264 | 279 | 310 | ||
Inosine | 252 | 262 | 263 | 320 | |
2’-Deoxyinosine | 252 | 263 | 320 |
Actual [%] | Predicted [%] | Difference [%] (UV/Vis-Assay - HPLC) | |
---|---|---|---|
HPLC | UV/Vis Assay | ||
0 | 0 ± 0 | 1.47 ± 1.02 | +1.47 |
20 | 21.08 ± 0.02 | 20.47 ± 0.25 | −0.60 |
40 | 40.89 ± 0.15 | 39.46 ± 0.09 | −1.43 |
60 | 60.03 ± 0.05 | 58.99 ± 0.38 | −1.04 |
80 | 79.38 ± 0.19 | 78.91 ± 0.13 | −0.46 |
100 | 99.67 ± 0.36 | 99.85 ± 0.15 | +0.18 |
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Kaspar, F.; Giessmann, R.T.; Krausch, N.; Neubauer, P.; Wagner, A.; Gimpel, M. A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases. Methods Protoc. 2019, 2, 60. https://doi.org/10.3390/mps2030060
Kaspar F, Giessmann RT, Krausch N, Neubauer P, Wagner A, Gimpel M. A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases. Methods and Protocols. 2019; 2(3):60. https://doi.org/10.3390/mps2030060
Chicago/Turabian StyleKaspar, Felix, Robert T. Giessmann, Niels Krausch, Peter Neubauer, Anke Wagner, and Matthias Gimpel. 2019. "A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases" Methods and Protocols 2, no. 3: 60. https://doi.org/10.3390/mps2030060
APA StyleKaspar, F., Giessmann, R. T., Krausch, N., Neubauer, P., Wagner, A., & Gimpel, M. (2019). A UV/Vis Spectroscopy-Based Assay for Monitoring of Transformations Between Nucleosides and Nucleobases. Methods and Protocols, 2(3), 60. https://doi.org/10.3390/mps2030060