Bioluminescence has become an increasingly important tool for researchers. It can monitor gene expression, track cell movements, and visualize tumor progression.
Currently, most bioluminescent systems use firefly or Renilla luciferases. However, the recent development of nanoluciferase (NLuc) provides improved stability and luminescence efficiency. This makes it ideal for use in preclinical applications.
Determine the Expression Level of mRNA
Although many cellular mRNAs have putative IRES sequences, most do not show apparent IRES activity when tested with the standard bicistronic mRNA test. This test involves inserting the IRES candidate into a bicistronic DNA plasmid with the Renilla luciferase (RLuc) reporter in the upstream position and firefly luciferase (FLuc) in the downstream position. The result is a molar ratio of RLuc and FLuc synthesis, determining IRES activity. However, this method has several limitations. It does not necessarily reflect the mRNA levels in cells, and it does not account for translation inhibition or mRNA degradation. It is therefore necessary to develop a better test for IRES function.
One such approach involves using monocistronic polyadenylated Appp-capped mRNAs. The test mRNA contains a very stable 5’-proximal hairpin and the candidate IRES. This approach avoids potential artifacts associated with plasmid DNA transfections and can be performed in the presence of wild-type or mutant mRNAs. It is also possible to use this approach in cell extracts from different tissues.
Another way to test IRES function is to use a cell-free system. The test mRNAs are capped and polyadenylated in vitro, and they contain a putative IRES and the downstream cistron of interest. Ideally, the mRNA should stimulate FLuc expression more than the expected bicistronic transcript. However, it is not easy to find a putative IRES that meets this criterion. Nevertheless, some IRESs do come close to meeting this requirement, including the Type I and II picornavirus IRESs.
Determine the mRNA Stability
A bioluminescent indicator, like Fluc, is a valuable tool for monitoring biological processes in cells or living organisms. It can be used to monitor cellular energy status and cell proliferation and to detect changes in the cytoplasmic distribution of enzymes and metabolites. It can also identify cellular signaling pathways, including the apoptotic pathway. In addition, it can be used to measure changes in cellular metabolism or protein expression.
However, it is essential to note that this assay may be somewhat sensitive. The reason is that many of the mRNAs expressed in the presence of a FLuc mRNA might have an unanticipated RLuc cistron at the 5′ end of the transcript. This cistron is not susceptible to siRNA-mediated silencing and might be translated. This could be true for mRNAs that are produced from cryptic promoters or that have undergone splicing.
The simplest way to overcome this problem is to perform the assay in nuclease-treated extracts. This method eliminates the possibility of competing translation from scanning-dependent mRNAs and is more reliable than using a siRNA against the anticipated full-length mRNA. Furthermore, it is an excellent idea to analyze the RNA recovered from these extracts by Northern blotting. This will help you to determine the exact amount of mRNA that is susceptible to silencing by the test mRNA.
Determine the mRNA Encapsulation
Compared to traditional mRNA quantification methods, the FLuc mRNA test provides a more detailed and comprehensive characterization of encapsulated mRNA. This approach yields information regarding the nucleic acid content of the mRNA-LNP complex in addition to quantitatively measuring mRNA encapsulation. The nucleic acid content of the mRNA-LNP can be determined either by comparing it to a standard curve prepared with a different sample or by analyzing the chromatogram or fractogram of the encapsulated mRNA-LNP mixture.
Bioluminescence imaging (BLI) is a non-invasive technique that uses a luciferin substrate to convert chemical energy into light. A luminometer then detects this light. The luciferin molecules are derived from bacteria and other organisms that contain a gene known as luciferase, which can convert a luciferin molecule into light. This light is produced by a process that involves enzymes, substrates, and oxygen.
The capacity of current mRNA quantification techniques to identify and describe monocistronic mRNAs is restricted despite their many benefits. This limitation is significant because monocistronic mRNAs can be very efficient IRES-dependent generators of RLuc expression, and a significant decrease in the relative ratio of FLuc to RLuc might indicate that IRES activity has been diminished by unanticipated splicing. Furthermore, IRES activity can be masked by the insertion of a very stable hairpin upstream of the tested luciferase cistron, which drastically reduces RLuc synthesis but may not prevent the generation of monocistronic mRNAs that can still demonstrate IRES-dependent RLuc expression.
Determine the mRNA Distribution
Many 5’-UTR sequences that are supposedly IRES-active promote a significant increase in FLuc expression, often several hundred-fold compared to the bona fide EMCV IRES control. These results are impressive at first glance, but they should be interpreted cautiously. Because these experiments use plasmid DNA transfection assays, they do not provide a direct comparison with the basal level of IRES-dependent translation observed in cell-free systems. Furthermore, the cryptic promoters that are likely to be responsible for these apparent IRES-dependent increases remain unknown.
Moreover, these cell-free systems are also prone to false-positive results, and they do not consider mRNA biodistribution. The biodistribution of LNPs in tissues can vary greatly depending on the mixing conditions. In addition, lipids used to synthesize LNPs can influence their biodistribution.
As a result, the most critical factor for predicting bioluminescence is the distribution of mRNA. This is why it is essential to study the mRNA distribution in a specific cell population or tissue. This can be done by comparing the distribution of mRNA in LNPs under different mixing conditions. This will help determine the best strategy for delivering mRNA to cells in a specific environment. Moreover, this technique will help scientists identify the factors involved in the bioluminescence process.
