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In-Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells




In-Vitro Transcription, Luciferase assay, Vaccinia virus, Poxvirus, Translation, 5’-UTR, 5’-poly(A) leader


In-Vitro Transcribed (IVT) RNA-based luciferase reporter assay enables studying the regulation of mRNA translation in poxvirus-infected cells. The assay can be used to study the translation regulated by cis-elements present in an mRNA, including 5’-untranslated region (UTR) and/or 3’-UTR.


Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5’-poly(A) leader in the 5’-UTR. In this study, to dissect the role of 5’-poly(A) leader in mRNA translation during poxvirus infection, we developed an in-vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises four core steps: (1) PCR to amplify the DNA template for in-vitro transcription; (2) In-vitro transcription to generate capped mRNA using T7 RNA polymerase and cap analog; (3) Transfection to deliver in-vitro transcribed mRNA into cells; (4) Detection of luciferase activity as indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5’-UTR and/or 3’-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be examined using this method.


According to the central dogma, genetic information flows from DNA to RNA and then finally to protein1,2. This flow of genetic information is highly regulated at many levels including mRNA translation3,4. Development of reporter assays to quickly measure regulation of gene expression will facilitate understanding of the regulatory mechanisms involved in this process. Here we describe a protocol to study mRNA translation using an in-vitro transcribed (IVT) RNA-based luciferase reporter assay in poxvirus-infected cells.

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Poxviruses comprise many highly dangerous human and animal pathogens5. Like all other viruses, poxviruses exclusively rely on host cell machinery for protein synthesis6–8. To efficiently synthesize viral proteins, viruses evolved many strategies to hijack cellular translational machinery to redirect them for translation of viral mRNAs7,8. One commonly employed mechanism by viruses is to use cis-acting elements in their transcripts. Notable examples include Internal Ribosome Entry Site (IRES), cap‐independent translation enhancer (CITE), etc.9–11. These cis-elements render the viral transcripts a translational advantage by attracting translational machinery via diverse mechanisms12–14. Over 100 poxvirus mRNAs have an evolutionarily conserved cis-acting element in the 5’-untranslated regions (5’-UTRs): a 5’-poly(A) leader at the very 5’ ends of these mRNAs15,16. The lengths of these 5’-poly(A) leaders are heterogeneous and are generated by slippage of the poxvirus-encoded RNA polymerase during transcription17,18. We, and others, recently discovered that the 5’-poly(A) leader confers a translation advantage to an mRNA in cells infected with vaccinia virus (VACV), the prototypic member of poxviruses19,20.

The IVT RNA-based luciferase reporter assay was initially developed to understand the role of 5’-poly(A) leader in mRNA translation during poxvirus infection19,21. Although plasmid DNA-based luciferase reporter assays have been widely used, there are several drawbacks that will complicate the result interpretation. First, plasmids are able to replicate in VACV-infected cells22. Secondly, cryptic transcription often occurs from plasmid DNA18,23,24. Thirdly, VACV promoter-driven transcription generates poly(A)-leader of heterogeneous lengths consequently making it difficult to control the poly(A)-leader length in some experiments18. An IVT RNA-based luciferase reporter assay will circumvent these issues and the data interpretation is straightforward.

There are four key steps in this methods: (1) polymerase chain reaction (PCR) to generate the DNA template for in-vitro transcription (IVTn); (2) IVTn to generate mRNA; (3) transfection to deliver mRNA into cells; and (4) Detection of luciferase activity as indicator of translation (Figure 1). The resulting PCR amplicon contains the following elements in 5’ to 3’ direction: T7-Promoter, poly(A) leader or desired 5’-UTR sequence, firefly luciferase open reading frame (ORF) followed by a poly(A) tail.  PCR amplicon is used as the template to synthesize mRNA by IVTn using T7 polymerase. During IVTn, m7G cap or other cap analog is incorporated in newly synthesized mRNA. The capped transcripts are transfected into uninfected or VACV-infected cells. Cell lysate is collected at desired time after transfection to measure luciferase activities indicating protein production from transfected mRNA. This reporter assay can be used to study translation regulation by cis-element present in 5’-UTR, 3’-UTR or other regions of an mRNA. Furthermore, IVT RNA-based assay can be used to study different mechanisms of translation initiation including cap-dependent initiation, cap-independent initiation, re-initiation and internal initiation like IRES.



Note: Information about the Material/Equipment used in this protocol can be found in Table of Materials.

  1. Prepare DNA template by PCR for in vitro transcription
    1. Consider crucial characteristics when designing primers discussed in these literature25–27.
    2. Design primers to generate PCR amplicon containing following elements in 5’ to 3’ direction: extra nucleotides, T7-Promoter, poly(A) leader, firefly luciferase ORF and a poly(A) tail. Primers (Forward and Reverse) need to encompass all the additional elements not present in the template DNA (Figure 2A).
    3. Design another set of primers for internal control of transfection efficiency containing the following elements in 5’ to 3’ direction: extra nucleotides, T7 Promoter, a random 5’-UTR coding sequence containing Kozak sequence, renilla luciferase ORF and poly(A) tail.
    4. Design forward primer (5’-3’) that includes several extra nucleotides28, T7-promoter, poly(A) leader or desired 5’-UTR sequence and approximately 20 nucleotides corresponding to the 5’ end of the reporter gene’s ORF. Make sure the corresponding region in the primer is identical to the sense strand (+ strand) of the gene.


  • For long 5’-UTR, synthesize two DNA fragments: one with T7 promoter followed by long 5’-UTR and second with reporter gene’s ORF. Join these two fragments using overlapping PCR29.

1.5.  Design reverse primer (5’-3’) that includes a poly(A) tail and approximately 20 nucleotides corresponding to the 3’ end of the reporter gene’s ORF. Make sure the corresponding region in the primer is identical to the anti-sense strand (- strand) of the gene and a stop codon is present before the poly(A) tail.


  • The desired length of A’s in poly(A) leader or poly(A) tail can be customized in the primers. For example, to add 50 A’s in the poly(A) tail, the reverse primer should entail 50 T’s. Similarly, to add 20 A’s in the poly(A) leader, the forward primer should entail 20 A’s.
  • The length of the nucleotide corresponding to the reporter gene’s ORF (5’ or 3’ end) should be adjusted based on the annealing temperature (Tm).
  • The sequence of all elements can be found in Table 1.

1.6.  In a PCR tube, add the reagents in the following order:

1.6.1.      DNase free water:      38 µl

1.6.2.      2X-Q5 Master mix:     50  µl

1.6.3.      Forward Primer (10 µM):     4 µl

1.6.4.      Reverse Primer (10 µM):     4 µl

1.6.5.      Luciferase DNA Template (1-10 ng/µl):      4 µl

1.6.6.      Total:       100 µl


  • Other High-Fidelity DNA polymerase product will suffice and the amounts of individual components in the mixture should be adjusted accordingly.
  • DNA template should be confirmed with correct sequence.

1.7.  Use a standard 3-step (Denaturation, Annealing, Extension) PCR cycle to generate desired PCR amplicon:

1.7.1.      Initial denaturation 95 °C:    2 minutes   (1X Cycle)

1.7.2.      Denaturation  95 °C:    15 seconds

1.7.3.      Annealing   X °C:    30 seconds   (25X Cycle)

1.7.4.      Extension   72 °C:     T minutes 

1.7.5.      Final Extension  72 °C:     7 minutes   (1X Cycle)

1.7.6.      Hold   4 °C:     ∞


  • Annealing temperature X °C depends on the primer set being used.
  • Extension time T minutes depend on the PCR amplicon size and DNA polymerase used.

1.8.  Detect the PCR product by running aliquots (10%) of PCR reaction in 1% agarose gel electrophoresis (containing 0.1 µg/ml ethidium bromide) along with commercially available molecular weight standard. Visualize the gel under UV illuminator to determine the size of the PCR product.

1.9.  After determining the correct size of the PCR product, purify it by using a commercially available PCR purification kit.

1.10.                    Once purified, determine the concentration of DNA and A260/A280 ratio (Ideally 2.0, ~1.8 is acceptable).

1.11.                    Purified DNA can be stored at -20 °C or used for IVTn immediately.

  1. Generate mRNA by IVTn
    1. Synthesize RNA from PCR product in vitro, using an in-vitro transcription kit.


  • HiScribe T7 Quick High Yield RNA Synthesis Kit is used in this protocol. Other in-vitro transcription kits should work as well.

2.2.  In a microcentrifuge tube, add the reagents in the following order:

2.2.1.      DNase-RNase free water:    X µl

2.2.2.      NTP Buffer Mix (20 mM of each NTP):  2 µl

2.2.3.      Cap Analog (Stock 40 mM):    4 µl

2.2.4.      Template PCR Product (400 ng):   X µl

2.2.5.      T7-RNA polymerase Mix:               2 µl

2.2.6.      Total:       20 µl


  • Volume X µl depends on the concentration of the Template PCR Product.
  • Vaccinia Capping System can also be used to cap RNA sequentially after IVTn.

2.3.  Mix thoroughly and incubate at 37 °C for 2 hours.

2.4.  Proceed with the purification of the synthesized RNA using an RNA purification kit.

2.5.  Determine the concentration of RNA and A260/A280 ratio (Ideally ~2).

2.6.  The purified RNA can be stored at -80 °C.

  1. Transfect mRNA to cells
    1. Seed cells in 24 well plate (To be approx. ~80-90% confluent next day) and incubate overnight in the incubator at 37 °C and 5% CO2.
    2. Infect cells with VACV at a Multiplicity of Infection (MOI) of 5.
    3. After desired hours post infection (hpi) (we often transfect at 10-12 hpi).  Transfect mRNA (500 ng of total mRNA per well of 24 well plates) using cationic lipid transfection reagent as shown in Figure 3.
      1.    For one well of 24 well plates, mix 480 ng of firefly luciferase (Fluc) mRNA and 20 ng of renilla luciferase (Rluc) mRNA in one microcentrifuge tube. In another microcentrifuge tube add 1.1 µl of cationic lipid transfection reagent.
      1.    Add 55 µl of reduced serum media in both tubes. Mix and incubate in room temperature for 5 minutes.
      2.    After 5 minutes of incubation, add 55 µl cationic lipid transfection reagent containing reduced serum media in mRNA containing tube.
      3.    Mix thoroughly and incubate in room temperature for 15 minutes.
      4.    During the incubation, remove the cell culture media and add 400 µl of reduced serum media per well of 24 well plates.
      5.    After incubation, add 100 µl of the mix to one well of 24 well plates.
  1. Measure luciferase activities

4.1. Five-hour post-co-transfection of Fluc and Rluc mRNA, measure luciferase activity using

a Dual-Luciferase Reporter Assay System (DLAS).

4.2  Remove reduced serum media and lyse the cells by adding 150 µl 1X Passive lysis buffer, a component of DLAS.

4.3.      After 10 minutes incubation at room temperature, collect the lysate by scrapping the cells and transfer to microcentrifuge tube.

4.4.      Centrifuge the lysate at 12,000 X g for 10 minutes at 4 °C to pellet cell debris.

4.5.      Add 30 µl of supernatant in opaque-walled 96 well white assay plate with a solid bottom.

4.6.      Measure the dual luminescence using DLAS in multimode plate reader luminometer.

4.7.      The measurement is taken using kinetics function (all steps on a per-well basis) using the following settings:

Inject Luciferase Assay Substrate (Fluc):  30 µl

Wait / Incubation time:    2 sec

Luminescence Measurement (Fluc):   10 seconds

Stop & Glo Substrate (Rluc):    30 µl

Wait / Incubation time:    2 sec

Luminescence Measurement (Rluc):   10 seconds

4.8.      Export the luminescence reading data into an excel file.

4.9.      Analyze the data.


The four steps of IVT RNA-based luciferase reporter assay:  PCR to generate DNA template for IVTn, IVTn to generate mRNA, mRNA transfection, and luciferase measurement, can be seen in the schematic diagram (Figure 1). Designing of primers for both DNA templates (Fluc and Rluc) and the general scheme of overhang extension PCR is illustrated in the schematic (Figure 2A). After PCR, the correct sized PCR product is detected by agarose gel electrophoresis (Figure 2B). Subsequently, the PCR product is used as the template to synthesize RNA in-vitro (Figure 3A), which is then purified and transfected into cells using cationic lipid transfection reagent (Figure 3B).

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The IVT RNA-based luciferase reporter assay was developed to understand the role of 5’-poly(A) leader in mRNA translation during poxvirus infection. Using this assay, we tested the translation efficiency of a Fluc mRNA that contains a 5’-poly(A) leader (12nt)  in uninfected and VACV-infected cells. The Fluc value was normalized using Rluc value in both uninfected and VACV infected cells to determine the relative Fluc activity (i.e. Fluc activity/Rluc activity) (Figure 4A). The division of Fluc by Rluc normalized the transfection efficiency and RNA stability in a particular well. Using this analysis approach, we determined that 5’-poly(A) leader containing mRNA has a translational advantage during VACV infection (Figure 4B). The advantage in infected cells was not due to differential transfection efficiency or mRNA stability as the RNA level was similar in uninfected and VACV infected cells 5 hours post mRNA transfection19.


Figure 1: Schematic of the experimental procedure.  PCR is used to generate a DNA template with desired elements. mRNA encoding a luciferase reporter gene is synthesized in-vitro using a T7-RNA polymerase based system. A Firefly luciferase (Fluc) mRNA is co-transfected with a Renilla luciferase (Rluc) mRNA into uninfected or VACV-infected cells. Luciferase activities are measured using a luminometer with dual luciferase capability.

Figure 2: Primer design and PCR-based DNA amplification.  (A) Forward primer is synthesized to include 8nt upstream of the T7 promoter followed by a desired 5’-UTR and part of the 5’ end of the luciferase reporter gene, while the reverse primer includes a poly(A) tail and the 3’ end of the luciferase reporter gene. By overhang extension PCR using a plasmid template containing luciferase gene, a DNA template is generated. (B) DNA band of the desired size from PCR reaction was detected using 1% agarose gel electrophoresis.

Figure 3: mRNA synthesis and transfection.  (A) Schematic of in-vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5’-UTR of interest and the T7 promoter is used as a template.  The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5’ to 3’ direction. Once mRNA is 25-30nt long m7G cap is added using an anti-reverse cap analog, ARCA. (B) Schematic demonstrating the transfection of reporter mRNA into cells. Medium containing either the reporter mRNA or cationic lipid transfection reagent in separate tubes is allowed to equilibrate at room temperature for 5 minutes. The solutions are then mixed followed by incubation at room temperature for 15 minutes after which the RNA/transfection reagent mixture is added into cells in culture plates.

Figure 4: Increased translational efficiency of mRNA containing a 5’-poly(A) leader.  (A) Fluc mRNA containing a poly(A) leader in the 5’-UTR and Rluc mRNA with the Kozak consensus sequence in the 5’-UTR are co-transfected into cells. (B) Fluc mRNA with 5’-poly(A) leader was transfected in uninfected and VACV infected cells. Five-hours post-co-transfection, luciferase activity was measured using a luminometer.

Table 1. The sequence of different elements. The table contains the sequence of T7-Promoter, poly(A) leader, Kozak sequence, firefly luciferase ORF, renilla luciferase ORF, poly(A) tail element in reporter PCR amplicon.


All four core steps: PCR, IVTn, mRNA transfection and luciferase assay, are critical to the success of IVT RNA-based luciferase reporter assay. Special attention should be given to primer design, especially for the T7 promoter sequence. T7 RNA Polymerase starts transcription from the underlined first G (GGG-5’UTR-AUG-) in T7 promoter added before the 5’-UTR sequence. Although the transcription start site (TSS) starts from the first G at the 5’ end, decreasing the number of G’s less than three in T7 promoter region decreased the RNA yield/output from IVTn. During the experiment, we observed that gel purified DNA product was not the best for IVTn as both yield and quality of RNA are lower. To work around this complication, we only ran 5-10% of the PCR reaction in 1% agarose gel electrophoresis to determine the size and purified the rest 90-95%, using a PCR purification kit, to be used for IVTn.

The proposed method is suitable for use in different model systems with some modifications like the method of mRNA delivery, internal control to be used, a suitable time for translation, sample preparation and analysis of data. Currently, this main limitation of this method is that it is an in-vitro assay to quickly test translation regulation by cis elements. We would like to emphasize this method should be corroborated by other in-vivo experiments, if possible.

Compared to DNA modification, the roles of RNA modifications are less studied. However, with the discovery of enzymes that write, read and erase RNA modifications30–35, it is now possible to study the influence of RNA modification in gene expression. The IVT RNA-based luciferase reporter assay can be modified to incorporate different RNA modifications and used to test their effect on RNA translation. First, this method can incorporate different cap analogs that have various modifications30. Additionally, supplementing an internal RNA modifying enzyme during or after IVTn can incorporate internal RNA modification. Addition of a modification to cap 0, cap 1, and an internal RNA modification will provide a tool to study the role of these RNA modifications in translation.

The IVT RNA-based luciferase reporter assay has great potential and broad applications in understanding basic biology about RNA translation. Different mechanisms for the initiation of translation, including cap-dependent initiation, cap-independent initiation, re-initiation and internal initiation such as IRES can be studied using this method. On top of these advantages, this assay can be employed to test translation regulation by cis-elements at 5’-UTR and/or 3’-UTR in an mRNA. This assay uses PCR product rather than plasmid to avoid troublesome and lengthy cloning, consolidates transcription and mRNA capping in a single reaction, and utilizes conventional transfection and analysis to make IVT RNA-based luciferase reporter assay a user-friendly, quick, and straightforward method to study mechanisms of mRNA translation.


  1. Crick, F. H. On protein synthesis. Symp. Soc. Exp. Biol. 12, 138–163 (1958).
  2. Crick, F. Central Dogma of Molecular Biology. Nature 227, 561–563 (1970).
  3. Sonenberg, N., Hinnebusch, A. G. Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets. Cell 136, 731–745 (2009).
  4. Spriggs, K. A., Bushell, M., Willis, A. E. Translational Regulation of Gene Expression during Conditions of Cell Stress. Mol. Cell 40, 228–237 (2010).
  5. Shchelkunov, S.N., Marennikova, S.S., Moyer, R.W. Orthopoxviruses Pathogenic for Humans. Springer US (2005).
  6. Gale, M., Tan, S.-L., Katze, M. G. Translational Control of Viral Gene Expression in Eukaryotes. Microbiol Mol Biol Rev 64, 239–280 (2000).
  7. Walsh, D., Mathews, M. B., Mohr, I. Tinkering with Translation: Protein Synthesis in Virus-Infected Cells. Cold Spring Harb. Perspect. Biol. 5, a012351 (2013).
  8. Cao, S., Dhungel, P., Yang, Z. Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff. J. Virol. 91, e00071-17 (2017).
  9. Pelletier, J., Kaplan, G., Racaniello, V. R., Sonenberg, N. Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5’ noncoding region. Mol. Cell. Biol. 8, 1103–1112 (1988).
  10. Pelletier, J., Sonenberg, N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320–325 (1988).
  11. Guo, L., Allen, E., Miller, W. A. Structure and function of a cap-independent translation element that functions in either the 3′ or the 5′ untranslated region. RNA 6, 1808–1820 (2000).
  12. Simon, A. E., Miller, W. A. 3′ Cap-Independent Translation Enhancers of Plant Viruses. Annu. Rev. Microbiol. 67, 21–42 (2013).
  13. Marom, L. et al. Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES. RNA Biol. 6, 446–454 (2009).
  14. Liu, B., Qian, S.-B. Translational reprogramming in cellular stress response. Wiley Interdiscip. Rev. RNA 5, 301–305 (2014).
  15. Ahn, B. Y., Moss, B. Capped poly(A) leaders of variable lengths at the 5’ ends of vaccinia virus late mRNAs. J. Virol. 63, 226–232 (1989).
  16. Ahn, B. Y., Jones, E. V., Moss, B. Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5’ poly(A) leader on its early transcript. J. Virol. 64, 3019–3024 (1990).
  17. Schwer, B., Visca, P., Vos, J. C., Stunnenberg, H. G. Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5′ poly(A) leader. Cell 50, 163–169 (1987).
  18. Yang, Z., Martens, C. A., Bruno, D. P., Porcella, S. F., Moss, B. Pervasive initiation and 3′ end formation of poxvirus post-replicative RNAs. J. Biol. Chem. 287, 31050–31060 (2012).
  19. Dhungel, P., Cao, S., Yang, Z. The 5’-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation. PLOS Pathog. 13, e1006602 (2017).
  20. Jha, S. et al. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase. Nature 546, 651–655 (2017).
  21. Dai, A. et al. Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff. J. Virol. 91, e01858-16 (2017).
  22. De Silva, F. S., Moss, B. Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors. J. Virol. 2, 23 (2005).
  23. Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. F., Moss, B. Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing. Proc. Natl. Acad. Sci. 107, 11513–11518 (2010).
  24. Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. F., Moss, B. Genome-Wide Analysis of the 5′ and 3′ Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts. J. Virol. 85, 5897–5909 (2011).
  25. Lorenz, T. C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J. Vis. Exp. JoVE (2012). doi:10.3791/3998
  26. Dieffenbach, C. W., Lowe, T. M., Dveksler, G. S. General concepts for PCR primer design. PCR Methods Appl. 3, S30-37 (1993).
  27. Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J. PCR Protocols: A Guide to Methods and Applications. Academic Press (2012).
  28. Baklanov, M. M., Golikova, L. N., Malygin, E. G. Effect on DNA Transcription of Nucleotide Sequences Upstream to T7 Promoter. Nucleic Acids Res. 24, 3659–3660 (1996).
  29. Liu, Q., Thorland, E. C., Heit, J. A., Sommer, S. S. Overlapping PCR for Bidirectional PCR Amplification of Specific Alleles: A Rapid One-Tube Method for Simultaneously Differentiating Homozygotes and Heterozygotes. Genome Res. 7, 389–398 (1997).
  30. Akichika, S. et al. Cap-specific terminal N6-methylation of RNA by an RNA polymerase II–associated methyltransferase. Science (2018). doi:10.1126/science.aav0080
  31. Sun, H., Zhang, M., Li, K., Bai, D., Yi, C. Cap-specific, terminal N 6 -methylation by a mammalian m 6 Am methyltransferase. Cell Res. 1 (2018). doi:10.1038/s41422-018-0117-4
  32. Boulias, K. et al. Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome. [Preprint] bioRxiv 485862 (2018). doi:10.1101/485862
  33. Dominissini, D., Rechavi, G. N4-acetylation of Cytidine in mRNA by NAT10 Regulates Stability and Translation. Cell 175, 1725–1727 (2018).
  34. Wei, J. et al. Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm. Mol. Cell 71, 973–985.e5 (2018).
  35. Fu, Y., Dominissini, D., Rechavi, G., He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014)


energy and are been widely used as fuel in automobile and industries. The components of the bagasse were chemically characterized by measuring their dry weight. Table # represents the composition of dry sugarcane bagasse analysed in the present study compared with data collected from other research articles. The dissimilarities in composition of lignin and cellulose might be due to genetics variations, growing location, methods of harvesting, growing conditions and analytical procedures.

Table 1. Major component of sugarcane bagasse

Cellulose (%) Lignin (%) References
46 19.6 Present Study
40.57 25.93 (Zeng, Tong, Wang, Zhu, & Ingram, 2014)
25 16.2 (Dhabhai, Jain, & Chaurasia, 2012)
40 23 (Irfan, Gulsher, Abbas, Syed, & Nadeem, 2011)
45.4 23.4 (Pereira, Jacobus, Cioffi, Mulinari, & Luz, 2011)

As per the generated data, cellulose content in the bagasse was 46%, which was further reformed into accessible form for the saccharification enzyme. While the lignin constituted 19.6%, thus removal of lignin was carried out by the pre-treatment of bagasse for an efficient enzymatic hydrolysis.

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Cellulose Unit Activity

The AumEnzymes, India generously donated two commercial cellulases, Acid Cellulase and Neutral cellulase. The cellulase activity of Aspergillus terreus, acid cellulase, and neutral cellulase were compared in order to proceed for the optimization of saccharification phase. The International Unit for enzyme activities (IU) of all the three cellulases were based on the total cellulase activity and endoglucanase activity, determined by the CMCase assay and FPU assay respectively. Table# represents the FPU and CMCase activity presented by all the three enzymes. The data in the table# clearly concluded that all the three cellulase have negligible total cellulase activity, while they have a high amount of endoglucanase activity.

Table 2. Comparison of Cellulase Activity

Cellulase CMCase Activity (IU) FPU Activity (IU)
Aspergillus terreus 0.273 0.045
Acid 0.966 0.028
Neutral 0.223 0.000

Which might indicate that all the cellulases has endoglucanase activity but, the negligible exoglucanases activity resulted in considerable reduction in total cellulase activity. Since the Acid cellulase had relatively higher enzyme activity, it was further used as the saccharifying enzyme. The protein content in the Acid cellulose was found using the protein assay and it was found to be 67.67 μg/mg of Acid cellulase powder. The specific activity was 14.11 IU/mg of Acid cellulase, indicating that 14.11 μmol of sugar is released by 1 mg of Acid cellulase (protein) in one unit.

Optimization of alkaline

The statistical design used for the microwave assisted alkaline pre-treatment is a four factors (weight of bagasse, power of microwave in wattage, NaOH concentration and the exposure time period) system, the response of the pre-treatment was based on the cellulose composition and reduced lignin after the pre-treatment. The design summary is shown in the Table #.

Table 3. Design Summary

Study Type: Response Surface Runs: 21
Initial Design: Central Composite
Design Model: Quadratic
Factor Name Units Low Actual High Actual Low Coded High Coded Mean
A Bagasse g% 2.5 10 -1 1 6.25
B Microwave W 100 600 -1 1 350
C NaOH g% 1 5 -1 1 3
D Time minutes 5 10 -1 1 7.5
Response Name Units Analysis Minimum Maximum C.V % R2
Y1 Cellulose g% Polynomial 0 81.2 9.3 0.9679
Y2 Lignin Removal g% Polynomial 0 67.25 8.54 0.9735

The design was a set of 21 runs, combinations of four factor experimental design, based on the RSM and CCD (Tabel#). The RSM is mathematical based system to study the interactions between the factors, while the CCD enables us to deduce an optimal condition for the pre-treatment.

Table 4. Test design and results of response surface analysis

Factor 1 Factor 2 Factor 3 Factor 4 Response 1 Response 2
Std Run A:Substrate B:Microwave C:NaOH D:Time Cellulose Lignin Removal
g W g% minutes g% g%
16 1 6.25 350 3.0 11.7 76.8 48.14
15 2 6.25 350 3.0 3.3 59.2 42.7
6 3 2.50 100 5.0 5.0 55 44.35
21 4 6.25 350 3.0 7.5 72.3 46.7
8 5 2.50 100 1.0 5.0 48.5 35.38
10 6 12.56 350 3.0 7.5 74.6 42.7
13 7 6.25 350 -0.4 7.5 48.25 40
5 8 10.00 100 1.0 10.0 50.6 38.4
9 9 -0.06 350 3.0 7.5 0 0
19 10 6.25 350 3.0 7.5 71.2 46.8
20 11 6.25 350 3.0 7.5 79.5 50.3
2 12 10.00 600 1.0 5.0 56.2 42.7
4 13 2.50 600 1.0 10.0 59.98 48.25
3 14 10.00 100 5.0 10.0 60.6 52.1
11 15 6.25 -70 3.0 7.5 61 48.53
18 16 6.25 350 3.0 7.5 77.1 44.23
7 17 2.50 600 5.0 10.0 75.6 62.5
12 18 6.25 770 3.0 7.5 76.3 67.25
17 19 6.25 350 3.0 7.5 69.7 48.9
1 20 10.00 600 5.0 5.0 71.85 57.23
14 21 6.25 350 6.4 7.5 81.2 60.56

According to the table#, runs #17, #18 and # 21 had maximum lignin removals while the #1, #1 and#21 showed maximum retained cellulose. The quadratic polynomial equations describes the correlation between the significant coefficients i.e. p-value (Prob>F) less than 0.05 and is used to obtain the regression values of coefficients where only significant coefficients are considered. But since this model supports hierarchy, the insignificant coefficients were not omitted. This equation was used to derive the predicted responses for cellulose (equation 1) and lignin removal (equation 2)


Equation 2

The adequacy of the quadratic model for the experimental responses (cellulose Y1 and lignin removal Y2) was checked using the Analysis of Variance (ANOVA), which was verified using the Fisher’s statistical model (F-value). The table# shows the ANOVA for Y2 response.

Table 5. ANOVA result of quadratic regression model for lignin removal

Source Sum of Squares Mean squares F-value p-value (Prob > F)
Model 3411.23 14 243.66 15.74 0.0014 significant
A-Bagasse 911.65 1 911.65 58.88 0.0003
B-Microwave 175.22 1 175.22 11.32 0.0152
C-NaOH 541.91 1 541.91 35.00 0.001
D-Time 14.80 1 14.80 0.96 0.366
AB 3.88 1 3.88 0.25 0.6347
AC 3.14 1 3.14 0.20 0.6684
AD 0.86 1 0.86 0.06 0.8216
BC 4.67 1 4.67 0.30 0.6028
BD 534.56 1 534.56 34.52 0.0011
CD 2.48 1 2.48 0.16 0.7031
A2 955.51 1 955.51 61.71 0.0002
B2 362.14 1 362.14 23.39 0.0029
C2 74.46 1 74.46 4.81 0.0708
D2 3.95 1 3.95 0.25 0.6317
Residual 92.90 6 15.48
Lack of Fit 71.34 2 35.67 6.62 0.0539 not significant
Pure Error 21.56 4 5.39
Cor Total 3504.13 20

ANOVA of the regression model for lignin removal had 15.74 “F-value” which described that the model is significant and also defined that there is only 0.14% chance that a “Model F-value” this large could arise due to noise. Since the “p-value” 0.0014, lesser than 0.005, it indicates that the lignin removal is sensitive to the coefficients/factors in the model. In other words weight of bagasse (A), microwave exposure (B), NaOH (C), BD, A2 and B2 have strong influence on the lignin removal. The p-value 0.0011 for BD (B-coded for microwave, D-coded for time), indicates the strong mutual interaction between B and D in removal of lignin. The “Lack of Fit F-value” of 6.62 justifies that there are 5.39% chances that such large values of “Lack of Fit F-value” might occur due to noise, where lack of fit is an error that would occur when one of the factor is omitted from the process model. Another statistical measurement that is a signal to noise is the ‘‘Adequate precision’’. The desirable ratio is greater than 4, as such the Adeq Precision value is 20.22, this model can be used to navigate design space and further optimization. “Multiple correlation corfficient or R2” value denotes the correlation between observed and predicted values, i.e. if the value is closer to 1, it means better correlation. In this case the R2 value is 0.9735, indicating better agreement between experimental values and predicted values. The “coefficient of variation (CV)” indicates the degree of precision to which the experiments are compared. The lower reliability of the experiment is usually indicated by a high value of CV. In the present case the CV value is low (8.5%) indicates a good precision and reliability of the experiment. At the same time, “Adjusted determination coefficient (Adj R2)” was high specifies improved precision and reliability of the conducted experiments.

The 3D surface plot illustrated below (Figure#) shows co-operative effect of microwaves and NaOH on the removal of lignin. From the plot, it can be predicted that with rise the concentration of NaOH and high powered microwaves exposure a increased degradation of lignin was observed, maximum lignin removal is observed with 5% NaOH concentration and microwave irradiation with power of 600W. But the low power microwaves and NaOH concentrations had no substantial removal of lignin.

Figure 1. Co-operative effect of Microwaves and NaOH on lignin removal

The second response considered in the pre-treatment was the amount of cellulose retained (Y1) after the process. The ANOVA of quadratic regression model for cellulose retained after pre-treatment illustrated in Table # is a significant model as evident from the Fisher’s F-test value (12.91) with a very low probability value [(Prob > F) = 0.0165]. This also indicates that there is only 0.24% chance that the F-value occurs due to errors during the experiments. Among model terms A, C, BD and A2 are also significant with probability of 99%. The interaction between B and D significant effect on increase in cellulose retaining response. The goodness of fit of the model was checked by determination coefficient (R2). In this case, the value of the R2 (0.9676) indicates that only 3.24% of the total variation between experimental values and predicted values are not explained by the model. The value of the adjusted determination coefficient (Adj. R2=0.8929) was also high, at the same time a relatively lower value of the coefficient of variation (C.V. = 9.3%) which indicates model is significant and the conducted experiment is consistent and has a good precision. The level of noise that affected the model is also very low, i.e. 11.16% determined using the Lack of Fit F-value (3.99). The Adequate Precision (15.608) for this model is greater than 4, this suggests the model can be used for navigating the design space and optimizing the experiment.

Table 6. ANOVA result of quadratic regression model for cellulose concentration after pre-treatment

Source Sum of Squares df Mean Squares F-value p-value (Prob > F)
Model 6226.99 14 444.79 12.91 0.0024 significant
A-Bagasse 2782.58 1 2782.58 80.76 0.0001
B-Microwave 117.05 1 117.05 3.40 0.1149
C-NaOH 779.62 1 779.62 22.63 0.0031
D-Time 154.88 1 154.88 4.49 0.0783
AB 36.72 1 36.72 1.07 0.3417
AC 1.56 1 1.56 0.05 0.8387
AD 8.14 1 8.14 0.24 0.6441
BC 27.27 1 27.27 0.79 0.4079
BD 1626.88 1 1626.88 47.21 0.0005
CD 1.51 1 1.51 0.04 0.8414
A^2 2013.06 1 2013.06 58.42 0.0003
B^2 4.08 1 4.08 0.12 0.7426
C^2 54.52 1 54.52 1.58 0.2552
D^2 8.46 1 8.46 0.25 0.6379
Residual 206.74 6 34.46
Lack of Fit 137.67 2 68.83 3.99 0.1116 not significant
Pure Error 69.07 4 17.27
Cor Total 6433.73 20

Figure # is a 3D response surface plot generated for 6.25 g of bagasse and 7.5 minutes of treatment by the regression mode, illustrates the effect of microwave irradiation (B) and NaOH (C) variables and the interactive effects of each on the cellulose concentration. It can be observed that by increasing both factors B and C results in increased cellulose concentration. The shading on the graph indicates the NaOH concentration from 3% to 5% is adequate for increasing the cellulose concentration to 75% and above along with the microwave irradiation within range of 350 W to 600W. Which indicates that higher microwave irradiation favours lignin removal. This results in high power consumptions and charring of cellulose. To avoid the destruction of cellulose to an inaccessible substance, the treatment can be carried at lower power microwave irradiations under high pressures.

The two response models of microwave assisted alkaline pre-treatment have shown positive influence on the removal of lignin and increased cellulose in bagasse. Thus the statistical analysis is reliable to generate the optimal conditions required for pre-treatment, the optimum condition was predicted using numerical optimization.

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The optimal values selected were, 6.37 g of bagasse irradiated at 350 W in 5% NaOH solution for 8.87 minutes. The predicted cellulose concentration was 81.94% and 56.6% lignin removal. The figure # represents the graph obtained using the numerical optimization methods.

Figure 2. Co-operative effect of Microwave and NaOH on cellulose concentration

Figure 3. Counter plot for predicted values of Lignin removal and cellulose concentration at optimized condition

There was 48% loss in dry weight of the bagasse after pre-treatment at optimized conditions, which might be either due to removal of lignin or lost during the washing process after pre-treatment bagasse. The result was similar to the work done by (Farid, Noor El-Deen, & Shata, 2014).

Optimization of Saccharification

The pre-treated bagasse was washed and further used for saccharification using the Acid cellulase. The efficiency of saccharification is evaluated by the saccharification%, it is the ratio of sugar released and the amount of polysaccharide present in the bagasse. Thus the saccharification% was used as the response factor for the statistical design used to optimize saccharification. The saccharification% response was assessed as a function of pre-treated bagasse loading (A), Acid cellulase loading (B) and time of incubation (C). The design developed using RSM and CCD is summarized in the Table # below.

Table 7. Design Summary

Study Type: Response Surface Runs: 20
Initial Design: Central Composite
Design Model: Quadratic
Factor Name Units Low


High Actual Low Coded High


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