Methods and systems for the detection of ricin and other ribosome inactivating proteins
A device, method, and system for the detection of ribosome inactivating protein activity, including the ricin toxin, in a sample. According to one embodiment, the ribosome inactivating protein in the sample removes an adenine from a labeled DNA substrate to create an abasic site. An AP lyase can then cleave the DNA substrate at the abasic site, allowing the fluorophore located at or near one end of the DNA substrate and the quencher at or near the other end of the DNA substrate to spatially separate. Once the fluorophore and the quencher are sufficiently separated, the fluorophore will emit a fluorescence signal. Increasing fluorescence, indicating ribosome inactivating protein activity, will be monitored in real time using a detection system.
1. A method for determining whether an unknown sample includes a ribosome inactivating protein, comprising the steps of:
providing a reaction mixture containing an apurinic/apyrimidinic (AP) lyase and a nucleic acid substrate having a fluorophore attached to a first end of the substrate, a quencher attached to a second end of the substrate, and a region in the substrate between the first end and the second end that will form an abasic site in the presence of a ribosome inactivating protein, wherein the nucleic acid substrate is selected from the group consisting of SEQ. ID NO. 1, SEQ. ID NO. 2, SEQ. ID NO. 3, SEQ. ID NO. 4, SEQ. ID NO. 5, SEQ. ID NO. 6, SEQ. ID NO. 7, SEQ. ID NO. 8, SEQ. ID NO. 9, SEQ. ID NO. 10, SEQ. ID NO. 11, and SEQ. ID NO. 12;
adding the unknown sample to the reaction mixture;
incubating the unknown sample in the reaction mixture such that any ribosome inactivating protein in the unknown sample will form the abasic site in the nucleic acid substrate and the AP lyase will cleave the nucleic acid substrate at the abasic site; and
measuring any fluorescence from the sample to determine whether the sample includes ribosome inactivating protein.
2. The method of claim 1 , wherein said ribosome inactivating protein is ricin.
3. The method of claim 1 , wherein the nucleic acid substrate is selected from the group consisting of: dsDNA, ssDNA, RNA, oligonucleotides, chimeric nucleic acids, and combinations thereof.
4. The method of claim 1 , wherein said region in the substrate comprises a sarcin-ricin loop.
5. A method for determining whether an unknown sample includes a ribosome inactivating protein, comprising the steps of:
providing a reaction mixture containing an apurinic/apyrimidinic (AP) lyase and a nucleic acid substrate having a fluorophore attached to a first end of the substrate, a quencher attached to a second end of the substrate, and a region in the substrate between the first end and the second end that will form an abasic site in the presence of a ribosome inactivating protein;
adding the unknown sample to the reaction mixture;
incubating the unknown sample in the reaction mixture such that any ribosome inactivating protein in the unknown sample will form the abasic site in the nucleic acid substrate and the AP lyase will cleave the nucleic acid substrate at the abasic site; and
measuring any fluorescence from the sample to determine whether the sample includes ribosome inactivating protein;
wherein the AP lyase is Endonuclease VIII.