RIBOSOMAL ENGAGEMENT POTENCY ASSAY
Provided herein are methods for detecting and/or measuring mRNA associated with ribosomes in a cell. The method can be used to assess potency and other characteristics of an miRNA drug product. Also provided are related products, including cells and reagents.
1 . A cell line comprising an immortalized cell comprising an engineered ribosome.
2 . The cell line of claim 1 , wherein the engineered ribosome comprises a ribosome-detectable tag fusion protein.
3 . The immortalized cell line of claim 1 or 2 , wherein the immortalized cell is a hematopoietic cell, a hepatic cell, or a cancer cell.
4 . The immortalized cell line of claim 3 , wherein the cancer cell is a hepatoma cell, a Hep3B cell, a HepG2 cell, or a Huh7 cell.
5 . The immortalized cell line of any one of claims 1-4 , wherein the engineered ribosome is encoded by a DNA sequence integrated into the cell genome.
6 . The immortalized cell line of any one of claims 1-5 , wherein the engineered ribosome comprises a ribosomal protein L (RPL).
7 . The immortalized cell line of any one of claims 2-6 , wherein the ribosome-detectable tag fusion protein is selected from the group consisting of a hemagglutinin (HA) tag, a hexa-histidine peptide, and a flag tag.
8 . The immortalized cell line of any one of claims 2-7 , wherein the ribosome-detectable tag fusion protein is attached to the engineered ribosome at a large ribosomal subunit.
9 . A method to detect ribosome-associated mRNA, the method comprising:
immunoprecipitating an mRNA-ribosome complex to produce an mRNA-ribosome-antibody complex, and
detecting a presence and/or an amount of the mRNA in the mRNA-ribosome-antibody complex.
10 . The method of claim 9 , further comprising:
transfecting a cell with the sample mRNA;
lysing the cell to obtain a cell lysate; and
isolating the mRNA-ribosome complex from the cell lysate.
11 . The method of claim 10 , wherein the cell is transfected with a lipid nanoparticle comprising the mRNA.
12 . The method of any one of claims 9-11 , wherein the ribosome is an engineered ribosome containing a detectable tag.
13 . The method of claim 12 , wherein the ribosome comprises a ribosomal protein L (RPL) and wherein the RPL contains the detectable tag.
14 . The method of claim 13 , wherein the RPL is RPL22.
15 . The method of any one of claims 12-14 , wherein the detectable tag is selected from the group consisting of a hemagglutinin (HA) tag, a hexa-histidine peptide, and a flag tag.
16 . The method of any one of claims 12-15 , wherein the engineered ribosome containing the detectable tag comprises RPL22HA.
17 . The method of any one of claims 9-16 , wherein the antibody is a monoclonal antibody.
18 . The method of any one of claims 9-17 , wherein the antibody is attached to a support.
19 . The method of claim 18 , wherein the support is a magnetic bead.
20 . The method of claim 19 , wherein the magnetic bead is added in suspension.
21 . The method of any one of claims 18-20 , wherein the antibody is bound to the support via a linker.
22 . The method of claim 21 , wherein the linker is a protein A/G linker.
23 . The method of any one of claims 9-22 , wherein the detecting comprising performing a polymerase chain reaction (PCR) using the mRNA in the mRNA-ribosome-antibody complex.
24 . The method of claim 23 , wherein the PCR is a reverse transcription quantitative PCR (RT-qPCR).
25 . The method of claim 23 or 24 , wherein the PCR method measures an mRNA signal by detecting a signature sequence on the mRNA.
26 . The method of claim 25 , wherein the signature sequence is within a 3′UTR.
27 . The method of any one of claims 9-26 , wherein a quantitative value of the mRNA is determined.
28 . The method of any one of claims 18-27 , further comprising washing the mRNA-ribosome-antibody complex with a salt buffer.
29 . The method of claim 28 , wherein the salt buffer comprises KCl, EDTA, and/or MgCl2.
30 . The method of any one of claims 10-29 , wherein the cell is from the cell line of any one of claims 1-8 .
31 . The method of any one of claims 10-29 , wherein the cell is an immortalized cell.
32 . A method for quantifying ribosomal engagement of sample mRNA in an immortalized cell line transfected with the sample mRNA comprising isolating an engineered ribosome complexed with the sample mRNA from the immortalized cell line and using a PCR method to quantify ribosomal engagement of sample mRNA.
33 . A complex comprising a ribosome, a mRNA, an antibody, and a reverse transcriptase.
34 . The complex of claim 33 , further comprising a magnetic bead.
35 . The complex of claim 34 , wherein the magnetic bead is attached to the antibody.
36 . The complex of claim 34 , wherein the magnetic bead is added in suspension.
37 . The complex of claim 33 , wherein the antibody is bound to a support via a linker.
38 . The complex of claim 37 , wherein the linker is a protein A/G linker.
39 . A complex comprising an engineered ribosome containing a detectable tag, a mRNA, and an antibody.
40 . The complex of claim 39 , wherein the engineered ribosome comprises a ribosomal protein L (RPL) and wherein the RPL contains the detectable tag.
41 . The complex of claim 40 , wherein the RPL is RPL22.
42 . The complex of claim 40 , wherein the engineered ribosome containing the detectable tag comprises RPL22HA.
43 . The complex of any one of claims 39-41 , wherein the detectable tag is selected from the group consisting of a hemagglutinin (HA) tag, a hexa-histidine peptide, and a flag tag.