Table 1.
Summary of depurination assays.
| Method Name | Assay System | RTA Sensitivity (Substrate) * | Detection Method | Advantages | Disadvantages | References |
|---|---|---|---|---|---|---|
| Aniline cleavage | In vitro | Rib: 1 ng rRNA: 10 ng |
Polyacrylamide gel separation. | First depurination method. Demonstrated mechanism of action of RIPs. |
Labor intensive. Susceptible to RNase contamination. Not adaptable for HTS. |
[9,11,16,49] |
| Primer extension | In vitro In vivo |
Rib: 10 ng | Polyacrylamide gel separation. Quantification by PhosphorImager. |
More accurate quantification with dual primer. Allows localization of the depurination site. |
Labor intensive. Susceptible to RNase contamination. Uses radioactive labeling. Not adaptable to HTS. Requires knowledge of the depurination site for primer design. |
[20,38,50,53] |
| qRT-PCR | In vitro In vivo |
Rib: 0.18 ng rRNA: 180 ng |
qRT-PCR with either absolute quantification or relative quantification by calculating fold change compared to a control. | Highly sensitive. Accurate quantification. Does not require radioactive material. |
Requires knowledge of the site of depurination for primer design. | [34,56] |
| HPLC | In vitro | SRL: 100ng | Adenine derivatization followed by HPLC or direct adenine detection by HPLC-MS. | Highly sensitive. Allows calculation of enzymatic kinetics. Does not require radioactive material. |
Labor intensive and time consuming. Not applicable to in vivo studies. Expensive equipment. Not adaptable to HTS. | [57,61,62] |
| Enzyme-coupled adenine detection | In vitro | Rib: 1.5 ng SRL: 60 ng |
Adenine conversion into readable colorimetric shift signal or luciferase light signal quantified by a microplate reader. | Fast and highly sensitive. Allows continuous measurement of depurination. Does not require radioactive material. Adaptable to HTS. |
High background if ingredients not pure. APRTase, PPDK and PRPP can be hard to obtain. Small molecules may interfere with enzymes in inhibitor screens. Saturating ribosome concentrations are hard to obtain. Not applicable to in vivo studies. | [34,36,64,65] |
* Rib: purified ribosome. α-sarcin/ricin loop (SRL): An RNA mimic of SRL. Abbreviations: RTA: ricin toxin A chain; RIP: Ribosome-inactivating protein; HTS: high throughput screening; PPDK: Pyruvate orthophosphate dikinase; PRPP: 5-phospho-α-d-ribosyl-1-pyrophosphate.