Structural elucidation of mRNA(Sirt1)- microRNA 34a complex
Mona Farshchian
Master thesis in Technology and learning, degree project for the study program Master of Science in Engineering and of Education, Stockholm
2015.
Degree Project in Technology and Learning of 30 ECTS in the program Master of Science in Engineering and of Education, Degree Program in Mathematics and
Chemical Science, Royal Institute of Technology, KTH, and Stockholm University, SU
Mona Farshchian: Structural elucidation of mRNA(Sirt1)-microRNA 34a complex, Stockholm 2015
MAIN SUPERVISOR
Peter Savolainen, Associate Professor, BIO, Kungliga Tekniska Högskolan
SECONDARY SUPERVISOR
Åsa Julin-Tegelman, Assistant Professor, Education, Stockholm University
EXTERNAL SUPERVISOR
Katja Petzold, Assistant Professor, Medical Biochemistry and Biophysics, Karolinska Institutet
EXAMINER
Joakim Lundeberg, Professor, BIO, Kungliga Tekniska Högskolan
Abstract
The aim of this thesis is to understand RNA-RNA interactions steering cellular functions, as in the case of this thesis the structure of mRNA(Sirt1) in complex with microRNA-34a (miR-34a). MiR-34a regulates the cancer protein p53 via Sirt1 modulation. This work will be the basis for future drug design and the understanding of misguided regulation in cancer.
The miR-34a binds to the mRNA(Sirt1) 3’ untranslated region (3’-UTR) and will either inhibit the translation of the protein Sirtuin 1 by capturing its mRNA or by degrading it.
p53, a key activator of miR-34a, prevents cancer development by inducing programmed cell death (apoptosis) on cells with DNA damage. In contrast, the protein Sirtuin 1 (Sirt1) has been shown to help cells with DNA damage to survive by down regulating the activity of protein p53 and will therefore increase the risk of cancer development. Studying the interaction between the mRNA(Sirt1) and miR-34a can present valuable information on the structure of the complex as well as the mode miR-34a uses to inhibit translation of mRNA(Sirt1) leading to down regulation of protein Sirtuin 1 and therefore prevent cancer development.
For the elucidation of this question different biochemical and biophysical methods were applied, such as in vitro transcription, gel electrophoresis, RNA purification with gel, crush
& soak and Cicular Dichroism (CD) melting studies. For this thesis work, the target sequence in mRNA(Sirt1) was optimized and purified so melting studies could be carried out. For future structural characterization using Nuclear Magnetic Resonance (NMR) studies with the miR-34a also produced in the lab.
The mRNA(Sirt1) target sequence was produced and purified with the final yield of 0.02%.
The results show that the sequence is highly ATP dependent and suggest the ratio between the nucleotides ATP/CTP to be 1:2. Low yield of purified mRNA(Sirt1) was received and still contained some impurities, which imply that another method than crush & soak should be used when purifying. The results, indicate that High-Preformance Liquid Chromatography (HPLC) might be a better solution for the pufication process.
The melting profiels done on mRNA(Sirt1) show that the secondary structures decrease with an increase in temperature. Accroding to the results, the mRNA(Sirt1) sequence is folded in room temperature, though not very stable. The wavelength which provided the best resolution was at 268 nm and the melting point of mRNA(Sirt1) was determined to 44
°C.
This thesis also contains an educational part, where an educational material was provided and testing was conducted for the subject Chemistry 2 for students age 18 and the material was evaluated with qualitative methods together with pedagogical methods. The study showed that the student can develope the different abilities stated in the curriculum with the material created. The results also showed that the students preferably choose cultural arguments when dicussing socio scientific question, rather than economical, democratic or utility arguments.
Keywords: mRNA(Sirt1), miR-34a, in vitro transcription, gel electrophoresis, CD spectroscopy, NMR, cancer regulation via p53, HPLC
Sammanfattning
Syftet med studien är att förstå RNA-RNAinteraktioner som styr cellulära funktioner, i detta fall mRNA(Sirt1) i komplex med microRNA-34a (miR-34a). MiR-34a reglerar cancerproteinet p53 via modulation av Sirt1. Detta arbete kommer lägga grund för framtida läkemedelsdesign vid reglering av cancer.
MiR-34a binder till den 3’ otranslerade regionen i mRNA(Sirt1) och hämmar antingen translationen av protein Sirtuin 1 (Sirt1) genom att fånga dess mRNA eller genom att försämra det. p53 förhindrar utvecklingen av cancer genom att framkalla programmerad cell död (apoptosis) av celler med skadat DNA. Det har visats att proteinet Sirtuin 1 hjälper celler med skadat DNA att överleva, genom att sänka aktiviteten av p53. På så vis ökar risken för utveckling av cancer. Genom att studera interaktionen mellan mRNA(Sirt1) och miR-34a kan värdefull information kring komplexets struktur fås. Samt hur miR-34a hämmar translationen av mRNA(Sirt1), vilket leder till minskad aktivitet av protein Sirt1.
För att klarlägga denna fråga har olika biokemiska och biofysiska metoder använts, såsom in vitro transkription, gelelektrofores, RNA rening med gel och Circular Dichroism (CD).
För detta arbete har målsekvensen i mRNA(Sirt1) optimerats och renats så CD smältstudier med kunde genomföras.
Resultatet visar att mRNA(Sirt1) sekvensen renats med ett utbyte på 0.02 %. Sekvensen är beroende av ATP och förhållandet mellan ATP/CTP nukleotider bör vara 1:2. Resutatet visar på ett lågt utbyte som visar på att High-Performance Liquid Chromatography (HPLC) kan vara en bättre metod än Crush & soak för reningen av mRNA(Sirt1).
Ur de smältprofiler som gjorts visade det sig att de sekundära strukturerna av mRNA(Sirt1) minskade med ökande temperatur. I enlighet med resultaten visar det att mRNA(Sirt1) är veckat i rumstemperatur men är inte stabil. Den bästa upplösningen erhölls vid 268 nm och mRNA(Sirt1) har en smältpunkt runt 44 °C.
Detta arbete innehåller även ett utbildningskapitel, där ett utbildningsmaterial har skapats och testats på 18-åriga kemi 2 studenter i åldern 18 år. Materialet har utvärderats med hjälp av kvalitativa metoder tillsammans med pedagogiska metoder.
Studien visade att de flesta förmågorna för kemi 2 kan utvecklas med hjälp av denna typ samhällsfrågor i det naturvetenskapliga klassrummet (SNI-fall) förutom förmågan att planera och genomföra experiment. Det argument som eleverna helst väljer att använda då de diskuterar det skapade SNI-fallet är Kulturargument och det minst använda är Demikratiargument.
Nyckelord: mRNA(Sirt1), miR-34a, in vitro transkription, gel elektroforesis, CD spektroskopi, NMR, cancer regulering via p53, HPLC
Acknowledgements
I would like to start by thanking the Petzold lab for giving me the opportunity to write and complete my thesis. Special thanks to Katja Petzold and Lorenzo Baronti for their guidance and support throughout this project. I would also like to thank my family and my fiancé Peyman Eshtiagh for always supporting me and encouraging me towards my goals.
I would also like to thank my KTH supervisor Peter Savolainen and SU supervisor Åsa Julin-Tegelman for all the support, along with everyone else who contributed to this thesis.
Abstract 3
Sammanfattning 4
Acknowledgements 5
1. INTRODUCTION 8
1.1 Introduction and background 8
1.2 Research questions addressed in Masters Thesis 9
1.3 Abbreviations and terms 10
1.6 LITERATURE STUDY 12
1.6.1 The human cell 12
1.6.2 The transfer of genetic information 13
1.6.3 The transcription reaction 14
1.6.4 Cancer 16
1.6.5 MicroRNA-mRNA interactions 16
1.6.6 MicroRNA 17
1.6.7 Sirt1-p53 regulation 18
1.6.8 Circular Dichroism 20
2. METHOD 21
2.1 Introduction 21
2.2 RNA by In Vitro Transcription 21
2.2.1 Annealing reaction 21
2.2.2 in vitro transcription by T7 - optimization 22
2.3 Polyacrylamide Gel Electrophoresis 25
2.3.1 Gel staining with Ethidium Bromide & UV detection 26
2.4 Large Scale Transcription reaction 26
2.5 mRNA(Sirt1) purification 27
2.6 Circular Dichroism UV melting studies 29
3. RESULTS & Discussion 31
3.1 Optimization of mRNA(Sirt1) 31
3.2 Large scale transcription reaction of mRNA(Sirt1) 35
3.2.1 Verifying with RNAse inhibitor and labeled nucleotides 35 3.2.2 Verifying with Pyruvate Phosphates and longer reaction time 36
3.2 Purification 36
3.4 CD melting studies of mRNA(Sirt1)-miR-34a complex 39
4. CONCLUSION 42
5. FURTHER RESEARCH 43
6. UTBILDNINGSKAPITEL 44
6.1 Inledning 44
6.2 Litteraturstudie 45
6.2.1 Forskning i klassrummet 45
6.2.2 Ämnesplaner skolverket 46
6.2.5 Samhällsfrågor med naturvetenskapligt innehåll 47
6.3 Syfte och frågeställning 47
6.4 Metod 48
6.4.1 Datainsamling 48
6.4.2 Urval och kvalitetsredovisning 48
6.5 Resultat 49
6.5.1 Förmågor som tränas med hjälp av SNI-fallet utifrån elevperspektiv 49 6.5.2 Förmågor som tränas med hjälp av SNI-fallet utifrån lärarperspektiv 50 6.5.3 Argument som elever använder i diskussion om frågan genmanipulation med hjälp av
SNI-fallet 50
6.6 Analys & Diskussion 52
6.7 Slutsats 53
6.8 Vidare forskning 53
7. REFERENCE 54
7.1 Articles and books 54
7.4 Graphics 57
8. APPENDIX A - Lab protocol 58
9. APPENDIX B - Calculations 62
10. APPENDIX C - Educational material 65
Bilaga 1. Lärarhandledning 65
Bilaga 2 Enkätundersökning 70
Bilaga 3 Kvalitativ intervju 71
1. INTRODUCTION
This part of the thesis explains the purpose and the background of the project, how it has been done and why the project has been carried out.
1.1 Introduction and background
This project is a master thesis in the field of Technology and Learning for the program Master of Science in Engineering and of Education at the Royal Institute of Technology and at Stockholm University. The project is in cooperation with the Petzold group at the Molecular Structural Biology unit at the department of Medical Biochemistry and Biophysics at Karolinska Institute. This thesis will help us understand the miRNA-mRNA interactions steering cellular functions. As in this case specifically, regulating the cancer protein p53. It is the basis for future drug design and understanding misguided regulatory, using laboratory techniques such a Gel Electrophoresis and CD spectroscopy.
One of the major causes of deaths among humans today is cancer, a disease affecting every third person (Campbell & Farrell, 2009). As the need for a cure increases, the greater responsibility lies in the hands of research groups to understand the reason for cancer development. Current research has found that a specific protein, p53, is mutated in most human tumours. The main role of protein p53 is to prevent cancer development, by inducing programmed cell death(apoptosis) when DNA damage occurs (Campbell &
Farrell, 2009). A large number of factors affect the function of the p53 protein, among those microRNA-34a(miR-34a) via the protein Sirt1.
The protein Sirt1 deacetylates protein p53 and down regulates its activity, which allows DNA damaged cells to replicate and in some cases develop into cancer. But when miR-34a down regulates Sirt1, it enhances the activity of p53 (deactivation missing by Sirt1) and these DNA damaged cells will encounter apoptosis and cancer formation is prevented.
Recent discoveries show that miR-34a can control the expression of protein Sirt1 by binding to mRNA(Sirt1)’s 3’ untranslated region (3’-UTR), which contains the regulatory region of gene expression(Fig 1.8) (Fujita et al., 2008). Therefore, the main focus of recent studies is understanding the course of action of miR-34a when approaching its target (Misso et al., 2014). This thesis was conducted to help explain the interaction between microRNA-34a and mRNA(Sirt1) in further understanding of its role in the expression of protein p53.
This thesis also consist of an educational part where an educational material was produced and tested on a class of students age 18. Due to the decreasing grades in Swedish schools in especially science oriented subjects, it is important to pay attention to the way schools approach these issues (Sjöberg, 2010). One way of awakening students interests in science is by discussing socio-scientific issues. The aim of the educational part is to create dialogue in the classrooms, by creating a socio-scientific issue to engage the students in conversation and study the way they build up arguments to take stand in difficult social issues. The project was conducted to create a material about a socio-scientific issue and evaluate the type of arguments students apply to take stand in these question and analyze what abilities the student can develop, in respect to the curriculum for the chemistry 2 course.
Qualitative methods such as interviews and questionnaire were applied to evaluate the provided material.
1.2 Research questions addressed in Masters Thesis
Scientific part of the thesis:
•
What are the optimal conditions for in vitro transcription reaction, to yield the most mRNA(Sirt1) product?•
How to purify mRNA(Sirt1) for structural studies?•
Elucidate the secondary structure and the stability of mRNA(Sirt1) with Circular Dichcroism.Educational part of the thesis:
•
What type of argumentations does students use to discuss ethical issues?•
What abilities can be developed with this type of educational material according to some students and one teacher?1.3 Abbreviations and terms
CD Circular Dichroism is a method used for structural studies of small molecules such as proteins and nucleic acids.
DNA Deoxyribonucleic acid contains contains genetic information.
DTT DTT helps to create the environment required for the
transcription initiate.
GMP Guanosinemonophosphate is a nucleotide that is used as
a monomer in RNA.
HPLC High-performance Liquid Chromatography is a method
for separating components in a mixture so each
component can be identified.
MgCl2 Magnesium chloride is a cofactor which the active site of the DNA strand needs to be able to be transcribed.
miRNA MicroRNA is a small noncoding RNA, which functions in for example transcirptional and post-transcriptional
regulation of gene expression.
MiR-34a MicroRNA-34a is a microRNA that plays a key role in tumour suppression and control targets involved in the cell cycle, differentiation and apoptosis. Part of the miR-34 family.
mRNA Messenger RNA is a molecule that carry genetic
information from DNA to the ribosome so proteins can
be produced.
mSirt1 Sirt1 messenger RNA codes for the protein Sirtuin 1.
NMR Nuclear Magnetic Resonance is a method used to study the molecular physics and structures of molecules.
PEG PEG is a molecular crowder used in the in vitro transcription.
p53 p53 is a tumour suppressor protein that protects the genome by inducing apoptosis in cells with damaged
DNA.
RNA Ribonucleic Acid contains information about protein
building.
rRNA Ribosomal Ribonucleic Acid is a RNA component in the ribosome which is essential for protein synthesis of the
ribosome.
siRNA Small interfering RNA functions by causing mRNA to be
broken down after transcription and causes RNA
silencing.
snRNA Small RNA with the function to process the pre-
messenger RNA in the nucleus.
SP Spermidine is a compound found in the ribosomes, which
promotes transcription.
tRNA Transfer RNA with the main assignment to transport amino acids to the ribosomes essential for the protein
synthesis.
TBE buffer Tris/Borate/EDTA is a buffer solution and is often used
in gel electrophoresis of nucleic acids.
1.6 LITERATURE STUDY
This part of the thesis presents discoveries made in this field and is related to this research. In this section biophysical techniques such as Gel electrophoresis and Circular Dichroism will be introduced as well as cellular functions where these techniques were be applied to.
1.6.1 The human cell
The human body is built up by a large amount of building blocks. The most fascinating and most complex mechanisms take place in the cells. A fully grown human body contains billions of cells with different functions. The composition and the functions of cells are fairly complex, but they all consist of water, proteins, lipids, carbohydrates and nucleic acids. One type of nucleic acid is deoxyribonucleic acid (DNA) that contains all the genetic information used for development and functioning of living organisms. A membrane surrounds the cell and gives it protection, stability and transports substances in and out.
Inside the cell a viscous liquid, cytosol, surrounds the organelles and provides them with necessary compounds. The organelles inside the cell have specific functions and all organelles are energy dependent of adenosine triphosphate (ATP), which they are provided by the mitochondrion. The mitochondrion produces ATP from carbohydrates, fats and proteins that the human body receives from consuming (Campbell & Farrell, 2012). It is fascinating how the different organelles and substances in the cell interact with one and other in order to maintain the cell and prevent errors from occurring. How the genetic information in the cell is transformed has been discovered by many researchers and many Nobel prices have been distributed in this manner, but everything has not yet been discovered.
Fig 1.1 Illustration of an Eukaryotic cell. The nucleus contains genetic information in the form of DNA. The mitochondria creates energy (ATP), which the organelles need in order to function properly. The ribosomes are sites
where the production of proteins take place. The cell membrane protects the cell and the cytoplasm provides the organelles with important compounds (Wikimedia, 2015).
1.6.2 The transfer of genetic information
The nucleus contains genetic information in the form of nucleic acid called DNA. The mechanism over how genetic information transfers in the cell is still not completely determined by scientist. The flow of genetic information can be described by the central dogma shown in Fig 1.2. Here, the information from DNA is transcribed into RNA in the nucleus and from there it is transported to the ribosomes to be translated into proteins.
The reaction where the DNA is transcribed into ribonucleic acid (RNA) is called the transcription reaction and the reaction where proteins are produced is called the translation reaction.
Fig 1.2 The central dogma. Mechanism describing genetic information transfers in the cell.
Until recent research, it was thought that RNA only functions as a messengerRNA (mRNA), ribosomalRNA (rRNA) or transferRNA (tRNA). New discoveries show, that there are many more functions of RNA shown in Fig 1.3. These functions have not been described in detail so far. When DNA is transcribed, not only mRNA, rRNA and tRNA are produced, but in fact during the transcription reaction different types of RNA are transcribed such as mRNA, tRNA, rRNA, small nuclear RNA (snRNA), microRNA (miRNA), small interfering RNA (siRNA) and long non-coding RNA (IncRNA), etc. All of these types of RNAs exist and are important with different functioning.
Fig 1.3 The genetic information flow. Until recently, it was thought that mRNA, rRNA and tRNA are the only RNAs existing, but now it is known that other RNAs such as miRNA, snRNA and IncRNAs etc. are existing and are important. Several recent nobel prices were received in this subject (Nobelprize, 2015).
Research has shown that the genetic information from DNA does not only have one direction as in Fig 1.2, but the information flow is far more complex shown in Fig 1.3. It has also been shown that information from RNA can be transcribed into single-stranded DNA, which is a method used by retroviruses such as HIV (Campbell & Farrell, 2012).
When DNA is transcribed into RNA, some long non-coding RNAs (lncRNAs) are transcribed. lncRNAs are functional RNA molecules that are not translated. These perform a number of vital functions within the cell. Most of these lncRNAs participate/regulate either in the transcription or translation reaction. One type of RNA is microRNA(miRNA) that participate in transcriptional and post-transcriptional regulation of gene expression.
The miRNAs regulate gene expression by base pairing with complementary sequences within mRNA molecules, which usually results in gene silencing, similar to siRNAs. The mRNAs, which these miRNAs bind are prevented from translation or are degraded. To further understand the interaction between mRNA and miRNA, the transcription reaction where mRNA is produced needs to be describe in order to understand how miRNA can bind to mRNA and inhibit its function. Research has described that one specific miRNA, microRNA-34a (miR-34a), has a key role in targeting specific proteins and enzymes that induce or prevent reparation of damaged DNA. When damaged DNA is not repaired it can lead to an increased cell growth and in many cases cancer (Misso et al., 2014).
1.6.3 The transcription reaction
DNA and RNA are both nucleic acids. DNA contains the genetic information, which is used by the different forms of RNAs to produce proteins and preform different functions.
Between DNA and RNA there are some main differences. The sugars in DNA and RNA are shown in Fig 1.4, the ribose sugar in RNA contains one -OH group more than the deoxyribose in DNA, which makes the RNA less stable than DNA (Campbell & Farrell, 2012).
Fig 1.4 The sugars in DNA and RNA are shown and one difference is that RNA has one extra -OH group.
Further differences between DNA and RNA are the base pairing between the nucleotides.
DNA uses the nucleotides Adenine(A), Thymine(T), Cytosine(C) and Guanine(G) (Campbell & Farrell, 2012). In RNA Uracil(U) is used instead of Thymine(T) and Uracil lack a methyl group on its ring in comparison with Thymine, shown in Fig 1.5.
RNA DNA
Fig 1.5 The differences between DNA and RNA and the structures of each nucleotide is visualized (Schmoop, 2015).
The transcription reaction usually takes place in the nucleus where the supercoiled DNA strands are unwound and opened up. One of the DNA strands called the template strand is used for synthesizing the RNA. Shown in Fig 1.6, RNA polymerase creates a transcription bubble to open up the double-helix and initiate the transcription reaction. RNA polymerase can only transcribe from 3’ end to the 5’ end so the DNA strand is read from 5’ to 3’ end (Campbell & Farrell, 2012). The other DNA strand called the coding strand is the strand which RNA polymerase makes a copy of and this is done by base pairing ribonucleoside triphosphates (rNTPs) with the DNA template strand. This base pairing is done by RNA polymerase in the same way the coding strand is base-paired. The polymerase then connects the rNTPs to a polymer by releasing pyro-phosphates and creates a di-ester- phosphate backbone, which has the same sequence as the coding strand but with the difference that the T’s are replaced with U’s (Campbell & Farrell, 2012).
Fig 1.6 Shows the transcription reaction (Limbic lab, 2015).
The transcribed mRNA in Fig 1.6 is called pre-mRNA because it has to undergo three processes called capping, polyadenylation and splicing before it is mature and ready to
travel to the ribosomes for translation. A post-transcriptional process called splicing needs to be performed because the pre-mRNA strand can contain both introns and exons. The introns are removed and only the exons are linked together in the mature mRNA strand.
The pre-mRNA contains a triphosphate group on its 5’ end, and this group is replaced by a structure called cap. This 5’ cap which is added to the mRNA strand helps the ribosomes to recognize the mRNA in the translation reaction. On the opposite site of the pre-mRNA strand a string called poly(A)-tail, which contains adenosine mono phosphates is added.
This reaction is called the polyadenylation and is important for the stability of the mRNA on its way from the nucleus to the ribosomes. Fig 1.7 shows the different regions in the mature mRNA strand before it is transported to the ribosomes for the production of proteins (Campbell & Farrell, 2012).
Fig 1.7 The mature mRNA strand after undergoing the post-transcriptional processes.
From all the regions in the mature mRNA strand only the coding region will be translated into a protein, but recent discoveries show that the 3’-UTR, containing regulatory regions, influences gene expression and is the part of the mRNA sequence where miRNA binds and inhibits the mRNA to proceed in translation reaction and producing proteins (Lee et al., 2010). MiRNAs bind to mRNAs e.g to 3’-UTR through its seed region which is located between positions 2 and 8 from the 5’ end (Cloonan, 2014). The role of mRNA-miRNA interactions are important for studies because of its link to inhibition of cancer development.
1.6.4 Cancer
One of the main causes of human deaths is cancer, and therefore the interest in understanding this disease has grown increasingly (Campbell & Farrell, 2012). Cancer is characterized by abnormal cell growth. Usually normal cells do not grow to such extent.
When DNA damage has occurred a signal reaches the cell and tells it to stop growing and to start apoptosis, but that is not the case in cancer cells. Cancer cells continue growing despite DNA damage (Campbell & Farrell, 2012). This type of cells have the ability to spread and grow in the body and become difficult to remove and cure, this is one reason why cancer is so lethal. Tumour suppressor proteins are produced by many of the human genes, which induce apoptosis when DNA damage occur. One particular tumour suppressor protein is protein p53. p53 is a transcription factor, it activates DNA repair proteins when DNA is damaged. p53 binds DNA and activates several genes and if the damage is irreparable it promotes apoptosis. The main role of p53 is to slow down cell division and to promote apoptosis. In many human tumours mutations of the p53 has been found. This because p53 is not able to bind DNA as it normally would (Campbell &
Farrell, 2009). Recent studies has shown that miRNAs have a great role in human cancer, which has led to comprehensive amount of research on the role of microRNA in tumour genesis (Misso et al., 2014). Although, until today, little is known about the structure of microRNA-mRNA complexes.
1.6.5 MicroRNA-mRNA interactions
MicroRNA has been the focus of a lot of research to understand the functions of it and how it biochemically and biologically interacts with its different targets (Cloonan, 2014).
Cloonan describes that many human diseases are often linked to deregulation of microRNA.
”Several mechanisms have been reported as to how miRNAs exert their effect on the overall protein production from genes. The first is by interfering directly with protein synthesis, either at the point of initiation or during elongation. The second is by mRNA destabilization, where the poly-A tails of mRNA
are shortened, leading to a higher turnover of the mRNA product by degradation”
(Cloonan, 2014, pp.379 )
MicroRNA has two pathways to target mRNA and affect the protein production. When microRNA bind to mRNA it inhibits the protein expression through two pathways, suppression of translation and mRNA degradation (Pasquinelli, 2012). To study the interaction between microRNA and mRNA many biochemical methods are applied, due to the complexity of RNAs.
1.6.6 MicroRNA
It was only in 1993 that the first microRNA was identified, named lin-4. Today more microRNAs are known. One function of miRNAs is to imperfectly bind to mRNAs and inhibit their transcription (Campbell & Farrell). MicroRNAs are non-coding RNAs containing about 22 nucleotides that regulate gene expression and can be divided into two groups, oncogenic microRNAs and tumour suppressor microRNAs (Cloonan, 2014). The interesting microRNAs for this thesis are the tumour suppressor miRNAs, due to their ability to prevent cancer development.
”In 2007, several groups identified the members of miR-34 family as the most prevalent p53-induced miRNAs”(Rokavec et al., 2014, pp. 1)
The members of the microRNA-34(miR-34) family are miR-34a, miR-34b and miR-34c.
Among these three, miR-34a has a key role in tumour suppression and plays an important role in this thesis.
MiR-34a controls the expression of numerous target proteins, which are involved in cell cycle, differentiation and apoptosis. Many components are involved in inducing or preventing apoptosis such as the protein p53 and protein Sirt1, which makes the understanding of the development of cancer fairly complex. Several mRNAs have been shown to be direct targets of microRNA-34a, one example is the mRNA(Sirt1), which codes for the Sirt1 protein (Misso et al., 2014). The function of protein p53 has been shown by many researchers, and its correlation to miRNA-34 and mRNA(Sirt1).
”Tumor suppressor p53 transcriptionally regulates expression of microRNA-34a, which confers translation inhibition and mRNA degradation of genes involved in cell cycle control and apoptosis.”
(Fujita et al., 2008, pp. 114)
Understanding the complex processes between p53, Sirt1 and miR-34a is crucial for gene regulation of gene expression and induction of apoptosis. For this reason, the mechanisms of the interaction between protein p53 and protein Sirt1 has been explained below.
1.6.7 Sirt1-p53 regulation
Sirt1-p53 regulation describes the correlation between miR-34a, Sirt1 and p53. As previously described, tumour suppressors inhibit transcription of cells with damaged DNA by promoting apoptosis. In many human cancers a mutation of the gene, which encodes p53 has been found.
In addition, Sirt1, an protein that deacetylates proteins and is highly NAD+ dependent helps the DNA damaged cells to survive. Recent studies done on mice demonstrate that during time of stress upon the cells, increased levels of Sirt1 allows the cells to survive when the cells actually was supposed to preform self-destruction (Campbell & Farrell, s.710). Studies have shown that miR-34a suppression of protein Sirt1 strengthens protein p53s promotion of apoptosis and avoiding cancer development.
”Therefore, SIRT1 mediates the survival of cells during periods of severe stress through the inhibition of apoptosis.” (Misso et al., 2014, pp. 3)
Sirt1-p53 mechanism is shown in Fig 1.9, which describes the dependency of all the different factors in preventing and inducing apoptosis. The activity of the p53 protein is repressed by Sirt1, through post-transcriptional deacetylation of the p53 protein. The protein Sirt1 is a target of miR-34a, which means that miR-34a can repress the activity of Sirt1 by binding to mRNA(Sirt1)’s 3’-UTR and can therefore induce the activity of p53 (Rokavec et al., 2014). Yamakuchi et. al. (2009) used in silicao analysis to screen for target genes of miR-34a and found that the 3’ -UTR of Sirt1 has a miR-34 responsive element.
Fig 1.9: The protein Sirt1 deacetylates protein p53 and down regulate its activity, which would allow DNA damaged cells to replicate and in some cases develop into cancer. But when miR-34a down-regulates Sirt1, it
enhances the activity of p53 (deactivation missing by Sirt1) and these DNA damaged cells will encounter apoptosis and cancer formation is prevented. Protein p53 is a key activator of miR-34a which ultimately targets mRNA(Sirt1) and reduces the Sirt1 protein levels(Lee et al., 2010). This is called a negative feed-back
loop.
The discovery that clarified that microRNA-34a targets the messenger RNA for the protein Sirt1 to increase the activity of p53, gives reason to study the interactions between the miR-
34a-mRNA(Sirt1) complex so further understanding about the miR-34a repression of Sirt1 can be obtained. Furthermore, Yamakuchi. (2009) verified that the repression of Sirt1 is a post-transcriptional inhibition. Accordingly, the mRNA levels do not decrease when miR- 34a targets mRNA(Sirt1), instead the levels of the protein Sirt1 decreases.
Fujita et.al (2008) did inspection of nucleotide sequences of the Sirt1 3’-UTR using TargetScan, which uses algorithms for the miRNA complementary sites. They showed that the 3’-UTR of mRNA(Sirt1) is a potential binding site for miR-34a. Illustrated in Fig 1.10, the binding site of miR-34a on the 3’-UTR of mRNA(Sirt1) is shown.
Fig 1.10: Fujita et al. (2008) showed this schematic representation of potential miR-34a binding site within the mRNA(Sirt1)’s 3’-UTR.
Both Yamakuchi et al. and Fujita et al. studied the binding sites of the miR-34a on the 3’- UTR applying different method. Both came across the same part of the sequence as a target of miR-34a. Accordingly, this part of the mRNA(Sirt1) sequence is of importance for this thesis to study the miRNA-mRNA interaction. When miR-34a binds to mRNA(Sirt1) it binds imperfectly, but the part of miR-34a that binds perfectly to the mRNA(Sirt1) sequence is called the seed sequence (Lewis et.al., 2005). The part of the miR-34a that does not bind to mRNA(Sirt1) can create bulges, hairpins, non canonical and canonical structures etc. (Pugsli, 1989).
The Petzold Lab used the already known sequence of mRNA(Sirt1) that has been shown to be targeted by miR-34a and used MC fold to predict the different possible structures of mRNA(Sirt1), miR-34a and mRNA(Sirt1)-miR34a complex using Mcfold. The structures that acquires the lowest energy is the most favourable structure (Major & Parisien, 2008).
The lowest energy for the mRNA(Sirt1) is -15.61kcal, and for miR-34a it is -14.09kcal. In order for these two sequences to bind the energy of the mRNA(Sirt1)-miR-34a complex has to be much lower. Otherwise it is more favourable for mRNA(Sirt1) and miR-34a to create structures separately, and therefore the bound complex of the two RNAs will be more populated than the structures of each of the components alone. After optimizing the length of the sequence of the mRNA(Sirt1)-miR-34a complex, it was determined that the lowest energy required to form the complex was -58,63 kcal, which is a lot less than the lowest energy level of the miR-34a and mRNA(Sirt1) would form separately. Fig 1.11, illustrates some of the different conformations of the mRNA(Sirt1)-miR34a complex.
Accordingly, a python script was used to build the DNA template which codes for the mRNA(Sirt1) strand with respect to the structure (with the help of C. Fontana).
Fig 1.11 The different structures of mRNA(Sirt1)-miR-34a complex, expected from the Mcfold (Major &
Parisien, 2008) analysis is that the most structure will be as base-paired as the end of the of the stem-loop as and also exist in other conformations in smaller amounts.
Additionally, the mRNA(Sirt1) used for this thesis has two additional G’s at the 5’ end because the polymerase used works better with the 5’GG sequence. It was analysed not to change the structure of the complex when adding the additional 5’GG. One method used to experimentally analyse the secondary different structure of mRNA(Sirt1) is Circular Dichroism.
1.6.8 Circular Dichroism
Circular dichroism (CD) spectroscopy is a widely used method to study secondary structures in proteins and nucleic acids (Sosnick et al., 2000). Here, a beam of circular polarized light consisting of both left hand and right hand polarized light strikes the sample. When the light strikes the optically active sample the polarization changes and this change is detected as a CD signal. Circular Dichroism is the difference in absorption between the left and right circular polarized light (Atkins & Paula, 2010). This method offers good resolution and allows the computation of values of rotational strengths, which is important for the absorption of light by helical molecules (Brahms & Mommaerts, 1964).
Melting curves can be obtained, which is a profile over absorbance versus temperature. The intercept of the curve, the melting point (Tm) can be found. The melting temperature is highly dependent of the concentration of the RNA strands. This transition contains information on what molecules are in transition, for example from hairpin to coil or duplex to single strand. The most efficient wavelength for measurements of the melting curves varies between 240-280nm, which is the wavelength of the maximum absorption. The maximum absorption is also called the hyperchromicity and is the amount of denatured nucleic acids. Comparison of melting curves can be done by comparing the percentage of hyperchromicity at different wavelength to yield information about the compositions of the bases in the RNA strand structures that are melting (Pugsli & Tinoco, 1989).
2. METHOD
This part of the thesis will give a description over the methods used to optimize the yield of mRNA(Sirt1), purifying the RNA and studying structure of mRNA(Sirt1). Different methods such as In Vitro Transcription, Gel Electrophoresis, Large-scale transcription reaction, RNA purification by crush & soak method and CD melting studies were applied.
2.1 Introduction
To begin this thesis, an introduction to the lab was required, to give an understanding of how the laboratory work should be carried out, obtaining information on how to proceed in case of emergencies and how to handle dangerous chemicals was mandatory. An introduction to the work, which the Petzold lab does was necessary to receive a deeper understanding of the methods and regulations within the field of the study.
2.2 RNA by In Vitro Transcription
To be able to study the binding structure of mRNA(Sirt1)-miR-34a complex, miR-34a and mRNA(Sirt1) was required. The engineered sequence of mRNA(Sirt1) had to be optimized in order to obtain the most yield of product. With the use of in vitro transcription the best conditions for mRNA(Sirt1) production was determined. This method is the most efficient method used for RNA production (Beckert & Masquida, 2011). According to Weissman et al. (2013), in vitro transcription is the most effective method when synthesizing RNA molecules from a template DNA sequence that includes a promoter sequence T7, a bacteriophage, followed by RNA polymerase.
In this thesis the DNA template was engineered from the mRNA(Sirt1) sequence predicted with MC-fold and the DNA was already ordered. All reactions were carried out in vitro, which means in an artificial environment instead of in a real cell. Although this method yields large amounts of product, it also contains impurities due to the unwanted activity of the polymerase. Despite the impurities, this method is a fair analytical technique for structural studies with Nuclear Magnetic Resonance (NMR).
Using polyacrylamide gel electrophoresis (PAGE) and ethidium bromide for staining, the mRNA(Sirt1) product could be detected with UV light. The preparation and implementation of the in vitro transcription was done according to the already existing protocol of the Petzold lab, Appendix A - Lab protocol. MiR-34a, was also produced in the lab.
2.2.1 Annealing reaction
Before beginning the production of the engineered mRNA(Sirt1), it was necessary to anneal the T7 primer to the DNA strand coding for the mRNA(Sirt1). The preparation of the annealing reaction was important because the annealed DNA and T7 promoter was going to be used in the transcription optimization reaction to produce mRNA(Sirt1). When working with RNA it is important to work clean, using gloves so no RNase from the skin or clothes would come in contact with the working space. Otherwise, the samples and the working space would be contaminated, which would affect the work negatively. All reagents and materials used were RNase free and autoclaved to ensure sterilization, because RNase contamination degrades RNA (Jasinski et al., 2015).
According to Fig 2.1, the compounds were added to an eppendorf tube with a pipette.
Here, the double-distilled water (ddH20) and magnesium chloride (MgCl2) were added to the eppendorf tube first. The DNA strand and the T7 primer were added at the end, to ensure that the right amount of the other two compounds were added. This was done due to the DNA strand and T7 primer being the most expensive ingredients.
Reverse strand DNA
7,50µL
T7 DNA primer 7,50µL
H20 6,00µL
MgCl2(0,01M) 9,00µL
Fig 2.1 Substances required for the annealing reaction.
Carefully with the tip of the pipette, the compounds were mixed and the blend was incubated for 5 minutes on 95 °C. Quickly, after the incubation the tube was placed on ice for 30 minutes for the annealing reaction to take place.
The annealing reaction refers to the part where the T7 primer attaches to the DNA strand according to the base-pairing encoded in the sequence. The T7 primer consisting 18 nucleotides, anneals to the 46 nucleotide long DNA template strand. This is shown in Fig 2.2.
Annealing reaction
Fig 2.2 T7 primer annealing on DNA template strand example.
Further, the preparations of the transcription reaction started while the annealing reaction was occurring.
2.2.2 in vitro transcription by T7 - optimization
Furthermore, the calculations for the transcription reaction begun, using the already existing template with the different compounds, Fig 2.3, which shows how a template for the transcription optimization reactions could resemble.
Fig 2.3 Template table for the transcription optimization
In accordance with Fig 2.3, eight samples were prepared and the total volume of each sample was adjusted to 50 µL with ddH2O. Here, the so-called ”master mix” was prepared consisting of the compounds kept constant, in order to reduce the amount of error in pipetting. To be on the safe side the master mix was prepared for nine samples instead of eight, in case a pipetting error would occur.
Moreover, all of the compounds were mixed together and the correct amount of the master mix was added. All samples were preheated for 5 minutes at 37 °C. The preheating of the eight samples were done in the incubation apparatus and all tubes were put on a boat before letting it into the water.
The next step where the polymerase was added had to be done quickly and carefully, due to RNA polymerases temperature sensitivity. RNA polymerase should be kept cold at all time otherwise it will denature. Therefore, this step, had to be carried out smoothly. The tubes were taken out of the incubation before taking the polymerase from the freezer. The polymerase was added to all the tubes and carefully mixed using the tip of the pipette.
Further, the samples were incubated for two hours at 37 °C.
As described earlier, the transcription reaction is the reaction where the RNA polymerase starts to transcribe the DNA template into an mRNA(Sirt1) strand. RNA polymerase uses the nucleotides in the sample and adds them according to the DNA template strand. Here, the RNA polymerase replaces all Thymines with Uracil nucleotides creating the mRNA(Sirt1) strand.
Visualized in Fig 2.4, the production of mRNA(Sirt1) from the annealing reaction with the help of RNA polymerase
Sample nr H2O
0.5M Tr is
0.25M MgCl2
0.25M DTT
0.25M SP PEG
GMP (100mM)
ATP (100mM)
GTP (100mM)
CTP (100mM)
UTP
(100mM) DNA POLY
1 27,8 0 6 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
2 22,8 5 6 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
3 17,8 10 6 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
4 12,8 15 6 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
5 23,8 10 0 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
6 19,8 10 4 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
7 15,8 10 8 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
8 11,8 10 12 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3
Transcription reaction
Fig 2.4 Showing the different steps in creating the final mRNA(Sirt1) sequence.
It was necessary to create an optimal environment for the polymerase depending on the template mRNA(Sirt1) to yield the most product. To determine the best conditions for the polymerase the annealing- and transcription reaction was repeated several times. The verification of the optimal conditions included analyzing the frequencies of the four nucleotides in the mRNA(Sirt1) sequence. Here, the calculations are presented in Fig 2.5 and these were useful in the optimization of mRNA(Sirt1). The more frequent nucleotides were added in larger amount and the less frequent nucleotides were added in less amount.
5’-GGACACCCAGCUAGGACCAUUACUGCCA— 3’
Nucleotide
Percent in the sequence m(Sirt1)
(%)
ATP 28,57
GTP 21,43
CTP 35,71
UTP 14,29
Fig 2.5 The percentage of each nucleotide in m(Sirt1) sequence.
After each transcription reaction, the yield of mRNA(Sirt1) product was visualized with polyacrylamide gel electrophoresis (PAGE) where the gel was stained with Ethidium Bromides. Using UV light, the bands on the gel which represented the mRNA(Sirt1) could be detected.
2.3 Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis is a method to separate biomolecules more precise, than other analytical techniques such as density-gradient centrifugation (Loening, 1967). The mRNA(Sirt1) produced by in vitro transcription can be separated with gel electrophoresis, due to the polyacrylamide gel composition. The gel consists of small pores, which helps so the charged biomolecules can run through the gel and separate. Due to the pores in the gel, smaller molecules can travel faster through the gel and the separation can be viewed with UV light after the gel has been stained with Ethidium Bromide (Campbell & Farrell, 2012).
While the samples were incubating, the preparation of the gel begun so the samples could be run on the gel directly after the incubation. RNA degrades with time when contaminated with RNase, therefore the injections of the samples are preferred directly after the incubation (Köhrer & Domdey, 1991), in order to avoid degradation. According to the protocol, Appendix A-Lab protocol, the gel plates were washed and cleaned from impurities before adding the gel solution. The gel plates should be put together in line in order to obtain a symmetric gel. When the gel plates were in order, the gel solution was prepared according to Fig 2.6.
COMPOUND VOLUME
20 % Polyacrylamide solution
50mL
10 % APS 300µL
TEMED 30µL
Fig 2.6 The combination of samples used for the polyacrylamide gel.
Polyacrylamide is dangerous because it is a carcinogen so it had to be handled carefully.
The polyacrylamide solution was added to a beaker and a 10 % ammonium persulfate (APS) was added with a pipette. APS consist of free radicals and initiates the formation of the gel. Also, N, N, N’, N’-tetramethylethylenediamine (TEMED) was added to the beaker and the solution was mixed carefully with the tip of a pipette. Slowly, the solution was poured inside the gel setup in between the gel plates. A comb was added and the gel was left to polymerize for 30 minutes. The TEMED was added to stabilize the free radicals from the APS and help the gel to polymerize.
Further, the polymerized gel was setup in the gel box and the apparatus was filled with Tris/Borate/EDTA(TBE) buffer. The added 10X buffer was diluted to 10 % (1X) with ddH20 and was used to stabilize the pH of the system. Here, the comb was removed slowly and the wells were cleaned with a syringe using TBE buffer. The wells needed to be clean before injecting the samples to remove Urea. Furthermore, the gel was preheated for 30 minutes on 12W. It is important not to tighten the glass plates intensely, due to risk of breakage. The system should be monitored and the buffer level should be checked from time to time. The apparatus can be damaged if the system leaks.
While the gel was being preheated the samples were taken out from incubation and mixed with loading buffer. Here, 9µL of loading buffer, Brohmphenolblue, Ethylenediaminetetraacetic acid (EDTA) and Formamidethen was added to 8 new eppendorf tubes. From each sample 1 µl was added to the tubes with loading buffer. Each
eppendorf tube consisted of a total volume of 10 µL, loading buffer and sample. Before using the loading buffer it was important to vortex it because the EDTA falls to the bottom. One reference with the annealing DNA strand and T7 promoter was prepared, with 9 µL of loading buffer and 1 µL of annealing reaction.
In accordance with the protocol, the wells were cleaned with TBE buffer again and while cleaning the wells the samples were incubated 1 minute on 95°C, so all of the tertiary and secondary structures would denature. The samples were injected into the wells of the gel with a pipette which measured 9,5 µL of each sample. When injecting the samples into the wells it was necessary to be careful so no loading errors would occur. The gel was then left to run for two hours on 12W and was under control so everything would go smoothly.
After two hours the gel was taken out of the gel box and carefully put into the staining solution before analyzing it with UV detection.
2.3.1 Gel staining with Ethidium Bromide & UV shadowing
The staining solution used to stain the polyacrylamide gel was Ethidium Bromide. Here, the staining solution was prepared and kept in a hood. Ethidium Bromide is a mutant and a carcinogen compound and requires caution. According to the protocol, the staining solution contained 1L of ddH20 and 20 µL of liquid Ethidium Bromide. Double gloves were used to put the gel into the staining solution and the gel was left 10 minutes for staining. Ethidium Bromide is mostly used after gel electrophoresis for detecting DNA and RNA, due to its ability to insert into the spaces between the helical oligonucleotides and forms a fluorescent complex which is viewed with UV shadowing (LePeqt & Paoletti, 1967). Since Ethidium Bromide was used as a staining agent and absorbs the light in the UV range and emits in the wavelength for visible light between 390-700 nm.
Furthermore, the gel was washed with ddH20 water for 5 minutes. While the gel was in the water, the camera and UV-Vis equipment were prepared. Plastic wrap was put on top of the UV light where the gel was going to be placed. It was necessary to be gental with the gel when transporting it from the water to the camera, because it is very sensitive to breakage after the staining. The gel was put on the plastic wrap over the UV light and bubbles were removed before analyzing it with the UV light. The pictures were taken with different exposure times to receive the best signal intensity for the gel. Further, the gel was thrown in a highly contaminated waste basket. It was observed that the staining solution degraded with time and the Urea concentration increased. The staining solution was renewed from time to time.
To yield the most mRNA(Sirt1) product, these steps were repeated for each new transcription reaction. When the results showed coherent and the best conditions were obtained, the preparations for the large scale begun.
2.4 Large Scale Transcription reaction
The chosen condition from the optimization reactions had to be tested with RNase inhibitor an labeled nucleotides before scaling up the reaction volumes from 50 µL to 5 ml.
Here, the reactions were prepared in the same way as the optimization reactions with the difference that RNase inhibitor was added to one sample and 13C 15N-labeled nucleotides for NMR measurements were used in the other sample. The chosen condition was also prepared as a comparison with the two new samples. Further the samples were run on a gel and it was decided whether the large scale should contain RNase or not and if the combination of compounds produced mRNA(Sirt1) with the labeled nucleotides or not.
Two ordinary samples were prepared and incubated one for two hours and one overnight, to see if the yield of product would differ between the two samples. A test with pyruvate phosphates was done at the same time to see how it would affect the obtained product.
According to the protocol, the best conditions of the compounds were mixed in the same way as in the optimization reactions. The annealing reaction was prepared in two different tubes and added together after the annealing step, because the reaction might not react the same way as it would in the larger scale. Here, a 5 ml sample was prepared instead of a 50 µL as before. Therefore, all substances were added with a 100 times larger volume into a falcon tube. The sample was incubated at 37°C for four hours. Further, the transcription reaction needed to be purified and the technique used was Crush & Soak. A thick gel was created and the sample was run with gel electrophoresis and afterwards the mRNA(Sirt1) band was cut out from the gel and purified.
2.5 mRNA(Sirt1) purification
The sample was taken from incubation and centrifuged at 4900 rpm at 4°C for 30 minutes.
According to the protocol, Appedix A-Lab protocol, the sample was filtered with a 0,2µm millipore tube to remove the phosphate precipitate. Meanwhile, an amicon filter was cleaned and filled with 15 ml ddH20. The amicon was run in the centrifuge at the same speed and temperature as the sample. When working with an amicon filter it is important not to let the filter run dry, because it will loose its function. The amicon filter should be placed perpendicular to the rotation axis so the solution can be pushed through the filter.
Further, the filtered mRNA(Sirt1) sample was placed in the amicon filter and centrifuged down to a volume of 1 ml. The 1 ml mRNA(Sirt1) sample was added to a new eppendorf tube and 1 ml of loading buffer was added to it. Here, the sample together with the loading buffer was going to be added to the thick gel.
The preparation of the thick gel had already started while the mRNA(Sirt1) was being centrifuged. In accordance with Fig 2.7, the gel solution for the thicker gel was prepared and poured into the gel setup that was adjusted for a thicker gel. Plastic wrap was added around the comb before inserting it to the gel, so only one large well would be created.
Due to the thickness of the gel, the polymerization took 45 minutes. The gel was then preheated 1 hour on 18W.
Fig 2.7 The combination of samples used for the thick polyacrylamide gel.
COMPOUND VOLUME
20 % Polyacrylamide solution
100mL
10 % APS 600µL
TEMED 60µL
Before injecting the 2 ml mRNA(sample) with the loading buffer to the gel it was boiled for 5 minutes on 95°C. Accordingly, 2ml of the mRNA(sirt1) sample was loaded on the gel with a pipette and the gel was left to run for 3 hour at 18W. The gel was monitored to check that everything was in order.
Accordingly, the gel was carefully taken out of the gel setup and placed on a plastic wrap on the table where the UV apparatus was already prepared. An A4 paper was colored yellow and put underneath the plastic wrap and the gel. The yellow paper was added to increase the contrast of the bands on the gel for UV-shadowing. Here, the band that corresponded to the mRNA(sirt1) was cut and put in a beaker. Using a small syringe, the band was crushed and soaked in elution buffer consisting of 10 % v/v 5M NaCl and a 10
% SDS sterile-filtered solution. Approximately 15 ml of elution buffer was added to the beaker and the beaker was left in the fridge overnight so the mRNA(Sirt1) molecules could separate from the gel pieces.
The following day the liquid was carefully removed with a pipette from the debris so no gel pieces would be drawn up. The liquid was put in an falcon tube and 10 ml of elution buffer was added to the debris and put back in the fridge for 2 hours. In this way more mRNA(sirt1) could release from the gel pieces. This was repeated once more and all the collected liquid consisting the mRNA(Sirt1) was concentrated using a amicon filter and a centrifuge as described earlier. The final concentration of the concentrated mRNA(Sirt1) sample was 500 µl.
With a pipett the 500 µl of sample was collected and put into a falcon tube where 50 µl of NaAc (3M, pH=5.2) was added and 1500 µl of 100 % ice-cold Ethanol was added and put into the freezer overnight.
Furthermore, the sample was centrifuged for 2 hours so the precipitate would separate from the supernatant. The sample was taken out of the centrifuge slowly and carefully so no mixing of the sample would occur. With a pipette, the supernatant was removed and put into another falcon tube and marked in case some mRNA(sirt1) would follow. The precipitate was in the bottom of the falcon tube and the same amount as removed liquid of 70 % Ethanol was added. The falcon tube was centrifuged for another 2 hours and the supernatant was removed again so only the precipitate would be at the bottom of the falcon tube. Plastic wrap with holes was put on top of the falcon tube instead of the lid, so the rest of the ethanol could evaporate overnight in the freezer.
In accordance with the protocol, the mRNA(Sirt1) sample was taken out of the freezer and the RNA pellet was washed with 1ml ddH20. Afterwards, the temperature was raised to 95°C for five minutes before cooling it down on ice for 30 minutes so the RNA could unfold and fold back again. From the sample, 2 µl was removed for measuring absorbance and determining the concentration of mRNA(Sirt1) in the sample. The absorbance was measured with a Nano Drop, which is a UV-spectrophotometer used to quantify and asses the purity if RNA, DNA and proteins. Water was used as a blank and 2 µl of mRNA(Sirt1) was measured. The concentration of the sample was calculated according to Lambert-Beer law, Fig 2.8, where A is the absorption coefficient, l is the pathlength, 𝜀 extinction coefficient and c is the concentration in molar. The extinction coefficient was determined by inserting the mRNA(Sirt1) sequence in the oligo-analyzer at the webpage Integrated DNA Technologies (IDT). Complete calculations can be found in Appendix C- Calculations.
Fig 2.8 Lambert-Beer law used for calculating the concentration of the sample containing the produced and purified mRNA(Sirt1)
The mRNA(Sirt1) sample was removed to a clean amicon filter and concentrated to 300 µl.
Accordingly, 2ml of NMR buffer was added to the amicon filter containing the concentrated sample. The filter was centrifuged for 25 min before adding another 2ml of NMR buffer (pH=6.5), consisting of 15mM NaP, 25mM NaCl and 0.1mM EDTA. The mRNA(Sirt1) sample with the as centrifuged to a final volume of 250 µl. Here, a regular gel was run to detect if the mRNA(Sirt1) was purified with the help of ethidium bromide staining and UV light.
A second purification was done due to the fact that more than the mRNA(Sirt1) band appeared on the gel. According to the protocol, a new thick gel was prepared and the sample was purified again as described for the first purification. The absorbance was measured with the nano drop to analyze how much mRNA(Sirt1) was left in the sample after the second purification.
2.6 Circular Dichroism UV melting studies
To study how the structure of mRNA(Sirt1) changes, different Circular Dichroism (CD) studies were done. The CD apparatus was turned on and the nitrogen flow was opened and level was checked to approximately 1 bar. The spectrum manager tool was opened and adjusted the apparatus automatically for 5 minutes.
Meanwhile, the Peltier heater and the water cooling system was turned on. To start with, the temperatur was set to 25°C. While the temperature was adjusting, two 10 mm cuvettes were washed thoroughly. The mRNA(Sirt1) sample prepared from the second purification, was boiled at 95°C for 5 minutes and left on ice for 30 minutes to anneal again.
Accordingly, 800 µl of ddH20 was added to one of the cuvettes and a CD spectra at 25°C was taken to see that the system was working as it should.
Further, 800 µl of NMR buffer was added to a cuvette and measured at 25°C as a blank for proper background subtractions (Sosnick et al., 2000). Afterwards the sample was added to the cuvette. After the second purification the amount of sample was measured to 250 µl so 500 µl of NMR buffer had to be added to the cuvette with the sample in order to measure the CD spectra for mRNA(Sirt1) at 25°C. Furthermore, four different CD spectra was obtained at the temperatures 25°C, 50°C, 75°C and 82°C. At each temperature 10 scans were made so an average could be determined and plotted in a curve. These 10 scans were done to receive a more accurate curves.
These were used to determine the largest difference in signal between the different temperatures in order to determine the best wavelength for running the melting profiles of mRNA(Sirt1). To determine the best wavelength for the melting studies a diagram was plotted with the wavelength and the difference in signal for mRNA(Sirt1) at 25 °C and at 82°C. However, when the best wavelength was determined the start temperature was set to
90 °C and the stop temperature at 5 °C together with the best wavelength. Data was collected each 0.2 °C. Accordingly, the CD folding and CD melting curves were fitted with the Hill’s equation, Fig 2.9.
Fig 2.9 The Hill’s equation used for fitting the CD folding and CD melting curves.
Accordingly, a is the max amplitude of f(T), b is the Hill’s coefficient and Tm is the melting temperature(Rinnenthal et al., 2010).
