Structural Elucidation of Mrna(Sirt1)- Microrna 34A Complex

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.

10 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).

DNA RNA

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.

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

15 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 7,50µL DNA

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.

Sample 0.5M 0.25M 0.25M 0.25M GMP ATP GTP CTP UTP nr H2O Tris MgCl2 DTT SP PEG (100mM) (100mM) (100mM) (100mM) (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

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

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’

Percent in the Nucleotide 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 50mL solution

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 25 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. 26

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.

COMPOUND VOLUME

20 % Polyacrylamide 100mL solution

10 % APS 600µL

TEMED 60µL

Fig 2.7 The combination of samples used for the thick polyacrylamide gel.

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, � 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 29

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).

3. RESULTS & Discussion This part of the thesis will describe the results obtained from the optimization of mRNA(Sirt1), mRNA(Sirt1) purification and CD melting studies of mRNA(Sirt1)-miR-34a complex.

3.1 Optimization of mRNA(Sirt1) synthesis To obtain the best conditions where maximum yield of mRNA(Sirt1) was produced the transcription reaction had to be repeated. Parameters such as substances, incubation time and temperature had to be varied as described in the method. Complications encountered could be smaller and larger side bands, not enough separation between the bands on the gel, impurities or bad Ethidium bromide staining which made it difficult to analyze the gel under the UV light. Especially, the human factor must be taken under consideration where pipetting errors, loading errors and contamination might occur. The polymerase does not always transcribe the sequence completely, which might cause the impurities during the optimization reaction. These impurities show up on the gels as smaller molecules under the mRNA(Sirt1) band. The gels made were verified several times and the result showed to be coherent. A compilation of the best samples were made in a gel, Fig 3.2 and Fig 3.1 demonstrates the different parameters being varied in each sample. These results indicate that sample 8 contains the most mRNA(Sirt1) product and these conditions were chosen for the large-scale transcription.

sample 0.5M 0.25M 0.25M 0.25M GMP ATP GTP CTP UTP nr H2O Tris MgCl2 DTT SP PEG (100mM) (100mM) (100mM) (100mM) (100mM) DNA Poly 1 19,8 10 6 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3 2 19,8 10 4 6 0,4 0 0 1,5 1,5 1,5 1,5 0,8 3

3 9,8 10 4 6 0,4 7 3 1,5 1,5 1,5 1,5 0,8 3

4 12,8 10 4 6 0,4 4 3 1,5 1,5 1,5 1,5 0,8 3 5 15,3 10 4 6 0,4 4 3 1,5 1,5 1,5 1,5 0,8 0,5

6 16,3 10 4 6 0,4 4 3 1,5 1 1,5 1 0,8 0,5

7 15,3 10 4 6 0,4 4 3 1 1 3 1 0,8 0,5 8 16,6 10 4 6 0,4 4 3 1 1 2 1 0,5 0,5

Fig 3.1 Transcription template for the final gel with the best samples from previous gels, all values added were in µl. Sample 8 was chosen to be the best conditions for the large-scale transcription reaction.

Fig 3.2 This gel is a compilation of the best combination of compounds, Fig 3.2, throughout the optimization process. One can clearly see the increase of target mRNA(Sirt1) in condition 8 in comparison with the other conditions.

DNA from the annealing reaction was always loaded on the gel as a reference and it appears on top of the gel as one strong band with 46 nucleotides (nt). The T7 promoter with its 18 nt has migrated further down on the gel when separating and is visible as a weaker band. To receive maximum yield of product one main factor is the purity and quality of the DNA. One way of confirming that the DNA is free from containing DNases is to run it on a PAGE and stain it with Ethidium Bromide (Schenborn & Mierendorf, 1985). In Fig 3.2, it is clear that the DNA from the annealing reaction does not contain anything more than the DNA and the T7 promoter. In all samples, the band above the mRNA(Sirt1) also called the plus one band was produced and was not managed to be eliminated in any of the different optimization reactions, however we managed to reduce it compared to our target sequence somewhat (comparison condition 7 & 8). The plus one band was slightly reduced in sample 8 and the mRNA(Sirt1) band was a bit stronger. Further, each different variation of substances will be described thoroughly.

Tris & Mg2+ Accordingly, both Tris and Mg2+ are important for the transcription reaction to occur. Tris is added to control the pH and Mg2+ is added because it is a cofactor that the polymerase active site requires in order to function properly. As expected, no mRNA(Sirt1) was produced in the samples without Tris and Mg2+. The dependence of Tris and Mg2+ for the reaction was clear but to determine the ratio between the two substances gels were made. The results showed that the preferred amount of Tris was 10 µl and for Mg2+ it was between 2 µl and 5 µl. When the amounts added of Mg2+ and Tris exceeded these amounts either less product was obtained or the side bands would increase. Fig 3.3 illustrates the Tris and Mg2+ dependence, where sample 2 and sample 1 resemble sample 1 and sample 2 in Fig 3.2. This gel was chosen due to the difference between sample 1 and sample 2 is

32 much greater and easier to visualize than in the compilation of gels showed before. Both samples contain 10 µl Tris where sample 1 contains 6 µl Mg2+ and sample 2 4 µl of Mg2+.

Fig 3.3 Gel showing the difference in intensity between sample 2 (4 µl Mg2+) and sample 1(6 µl Mg2+) and the Tris (10 µl) was kept constant in both. The sensitivity and dependency of Tris and Mg2+ is clear.

PEG & GMP The mRNA(Sirt1) starts with the nucleotide G at the 5’ end of its sequence, so GMP was added to the transcription reaction with the expectance that GMP would influence the transcription reaction and increase the yield of product. PEG was added as a molecular crowder because polymerase functions better in an environment that resembles the cell. As expected, the bands grew stronger when adding GMP and PEG. This can be visualized in Fig 3.2 where sample 3 containing GMP and PEG has stronger bands than sample 1 and sample 2, which do not contain those compounds.

DNA & Polymerase Furthermore, the effect of DNA and polymerase was tested with the expectation to reduce the extra bands that show up underneath the mRNA(Sirt1) band. From Fig 3.2, the results indicate that the polymerase was very concentrated. This lead to a decrease in mRNA(Sirt1) product which is clear in sample 4 in comparison with sample 5 in Fig 3.2. Here, sample 4 consists of the same amount of DNA but six times less polymerase. This confirmed that the polymerase was very concentrated and that the yield of mRNA(Sirt1) product increased when decreasing the polymerase amount. DNA and polymerase are dependent on the nucleotides added to the reaction solution when creating mRNA(Sirt1) product. Therefore variations of the nucleotides were done.

ATP & CTP The variations of the nucleotides were done according to the frequency of the different nucleotides in the mRNA(Sirt1) sequence. According to Fig 2.5 where the percentage of each nucleotide in the sequence was calculated the mRNA(Sirt1) consists of mostly CTPs and ATPs. Therefore, the amounts of GTPs and UTPs were lowered and variations of ATPs and CTPs were done. The results determine that by increasing the amount of CTPs and lowering the amount of the other nucleotides the yield of mRNA(Sirt1) product will increase. The amount of DNA and Polymerase was showed to be most efficient when the same amount of the two was added. Fig 3.4 demonstrates the ATP dependence of the mRNA(Sirt1) sequence. Here, the amount of DNA and polymerase is 0,5 µl and the amount of CTP and UTP is 1 µl. The mRNA(Sirt1) was shown to be very ATP dependent.

Fig 3.4 Shows the ATP dependence of mRNA(Sirt1). The concentration of the other substances are kept constant as obtained through the optimization reactions. Here, 0,5 µl of DNA and 0,5 µl of polymerase is added. When the ratio between ATP and CTP is around 1:2 the most yield is obtained, but when the ratio increases or decreases the mRNA(Sirt1) looses intensity.

The results show that the best condition to produce the most mRNA(Sirt1) product is when the ratio between ATP and CTP is 1:2 and it is clear that the mRNA(Sirt1) band decreases in intensity when the ATP/CTP ratio increases or decreases 1:2. Accordingly, sample 2 in Fig 3.4 corresponds to sample 8 in Fig 3.2, which was the sample chosen to create the most mRNA(Sirt1) product.

What are the optimal conditions for in vitro transcription reaction, to yield the most mRNA(Sirt1) product?

The results show that the mRNA(Sirt1) is dependent on Tris and Mg2+ in order to function properly through the transcription process. Further, it is clear that the polymerase used for this transcription process functions better in the presence of PEG and GMP. GMP speeds up the reaction, because the engineered mRNA(Sirt1) sequence starts with GTPs. When lowering the amount of added polymerase and DNA the intensity of the side bands decreased, while the intensity of the mRNA(Sirt1) band increased. The sequence preferred the same amount of DNA and polymerase, due to the polymerase being used was highly concentrated. As expected, to optimize the yield of product it is important to adjust the amount of added nucleotides with respect to the frequency of the different nucleotides in the mRNA(Sirt1) sequence. Here, the amount of added CTPs were increased and the amount of the other nucleotides were decreased. The results also show that the mRNA(Sirt1) sequence is highly sensitive to the ATP amount. The best ratio between ATP and CTP was shown to be 1:2. The impurities appearing on the gel are probably due to polymerases unwanted actvity and uncomplete transcription reactions.

3.2 Large scale transcription reaction of mRNA(Sirt1)

Before starting the 5ml large-scale transcription it was necessary to test how the mRNA(Sirt1) would react to RNase inhibitor and labeled nucleotides as described in the method. RNase inhibitor is usually added to the large-scale transcription reaction to minimize the degradation caused by contamination with RNases, which can occur during the process and are unnoticed. With the use of RNase inhibitor mRNA(Sirt1) product can be increased. Further, the labeled nucleotides are added to prepare the sample for future NMR studies.

3.2.1 Verifying with RNAse inhibitor and labeled nucleotides Fig 3.5 illustrates the different compounds in each sample, where sample 1 is the best conditions determined from the optimization reaction, sample 2 contains RNase inhibitor and sample 3 is with labeled nucleotides. Here, Fig 3.6 shows the obtained gel from the test reaction.

Sample H2O 0.5M 0.25M 0.25M 0.25M PEG GMP ATP GTP CTP UTP DNA Poly (100mM) (100mM) nr Tris MgCl2 DTT SP (100mM) (100mM) (100mM)

1 17 10 4 6 0.4 4 3 1 1 2 1 0.5 0.5 Regular 2 17 10 4 6 0.4 4 3 1 1 2 1 0.5 0.5 RNase 3 17 10 4 6 0.4 4 3 1 1 2 1 0.5 0.5 Labled

Fig 3.5 Transcription template for verifying mRNA(Sirt1) product with RNAse inhibitor and labeled nucleotides.

Fig 3.6 Gel obtained from the verifying optimization of mRNA(Sirt1) (lane 1) with RNase inhibitor (lane 2) and labeled nucleotides (lane 3). The results show that the yield of mRNA(Sirt1) product is stronger with the RNase inhibitor and the labeled nucleotides.

The results show that the amount of mRNA(Sirt1) product increases in the presence of RNase inhibitor and the labeled nucleotides. Accordingly, RNAse inhibitor was added to the large-scale transcription. A test with pyruvate phosphates was also done in the same way to determine if the yield of mRNA(Sirt1) product would increase.

3.2.2 Verifying with Pyruvate Phosphates and longer reaction time Furthermore, a test with pyruvate phosphates was done in the same way as RNase inhibitor and the labeled nucleotides. Fig 3.7 shows the gel where sample 1 is the optimized reaction incubated for two hours, sample 2 is identical with sample 1 but was incubated overnight and sample 3 was an ordinary sample with pyruvate phosphates. Here, the incubation time was increased because the yield of product might be larger when the incubation time is more. But in the case of mRNA(Sirt1), the results show that the yield of product is decreases with time. Sample 3 containing the pyruvate phosphates has slightly stronger band than sample 1 without it. Accordingly, the large-scale transcription it was decided to add Rnase inhibitor and pyruvate phosphates. The incubation time was also set to four hours due to the amount being larger in the 5 ml transcription reaction.

Fig 3.7 Gel obtained from the verifying optimization of mRNA(Sirt1) at different times and with pyruvate phosphates. Sample 1: Ordinary 2 hour transcription reaction. Sample 2: Transcription reaction overnight. Sample 3: 2 hour transcription reaction with 0,5 µl pyruvate phosphates.

3.2 Purification During the purification a problem was encountered. Before precipitating the mRNA(Sirt1), all of the concentrated mRNA(Sirt1) was not collected for the ethanol precipitation. Therefore it led to three different NMR samples containing different concentrations of mRNA(Sirt1). These three samples were run on a gel shown in Fig 3.8 and from the gel it was clear that all samples contain mRNA(Sirt1). Calculations over the three samples concentrations was done, Appendix B-Calculations. Sample 1 had the concentration 0.97µM, sample 2 3.27µM and sample 3 0.55µM. Consistent with the concentrations, the bands vary in intensity and sample 3 which had the lowest concentration have weak bands. For assurance, both 1µl and 10µl was loaded on the gel in case of the samples being very diluted.

Fig 3.8 The three purified samples loaded on a gel with 1µl and 10 µl of each. Two strong bands are visible, which indicates that the mRNA(Sirt1) is not completely purified.

This gel seems to contain impurities because the bands are very smeared and not clear in comparison with the previous gels. Therefore, it is necessary to clean the glas plates thoroughly and make sure no water is left before polymerizing the gels. In Fig 3.8, it was difficult to see wether the mRNA(Sirt1) was purified or not. The three samples were added together and loaded on a gel. Here, the concentration of the sample was 4.97µM with a mass of 122µg mRNA(Sirt1) product. Accordingly, the yield after the first purification was 0.7%, Appendix B - Calculations.

Fig 3.9 All samples in Fig 3.8 was added and run on a new gel with different amounts. The intensity of bands increase as the amount of loaded material increases. Two separated bands are visible so the conclusion could be drawn that the mRNA(Sirt1) was not purified completely.

The results show that the mRNA(sirt1) is not purified well enough because two strong bands occur on the gel. One possible explanation to why the mRNA(Sirt1) sample still contained the plus one band is the difficulty when cutting the band. The method with Crush & Soak is not the most efficient method for the purification of RNA generally. A new method, high-preformance liquid chromatography promises to be a more effective

37 method, which was helped setting up in the lab during this thesis. However, it was decided to purify the sample once again. Fig 3.10 shows the gel after the second purification. To make sure no mRNA(Sirt1) got lost, the plus one band was also purified to ensure it did not contain any mRNA(Sirt1).

Fig 3.10 Gel after second purification. Those samples with a +1 written underneath is the samples containing the plus one band and the others are the mRNA(Sirt1) sample. 10µl of the mRNA(Sirt1) sample was loaded twice due to some loading error at first. Two weak bands are visible which indicate that the mRNA(Sirt1) is still not purified.

According to Fig 3.10, the mRNA(Sirt1) was still not completely purified. Two bands were still visible in the last well of the gel. By now the sample was very diluted, with a concentration of 0,33µM and the mass 2.77µg. The final yield was calculated, Appendix B- Calculations, to approximately 0.02%. This yield is relatively good after two purifications and the mRNA(Sirt1) being lost during the first purification process. To increase the yield one have to be very careful throughout the purification process both not to loose mRNA(Sirt1) product but also not contaminate the sample. CD melting studies was done on the mRNA(Sirt1) with CD spectroscopy.

How to purify mRNA(Sirt1) for structural studies?

According to the results, the production of mRNA(Sirt1) yield was larger when adding RNase inhibitor and pyruvate phosphates. The results show, that RNase inhibitor minimizes the contamination during the large scale transcription and pyruvate phosphates slightly eliminates the smaller products appearing under the mRNA(Sirt1) band. The amount of mRNA(Sirt1) is less when incubating overnight than after two hours, which shows that the most efficient incubation time is around two hours for 50µl transcription and for 5ml large scale transcription around four hours. The conditions chosen for the large scale transcription also seems accurate for the large scale transcription with the labeled nucleotides for future NMR analysis. The final yield after the second purification was 0.02% which is a relative amount after two purifications. A better alternative method for purifying mRNA(Sirt1) would be HPLC where less product would be lost.

3.4 CD melting studies of mRNA(Sirt1)-miR-34a complex

Fig 3.11, shows the melting profiles obtained with CD spectroscopy at the temperatures 25 °C, 50 °C, 75 °C and 82 °C. Although the NMR blank was subtracted at all temperatures, the melting curves are noisy. The sample used for these measurements was diluted which likely might have caused the bumps in the spectra. As expected, the highest signal was received around 260 nm at 25 °C. Here, most of the secondary structures appear and the intensity of the signals decrease when the temperature increases. This is due to the secondary structures being destroyed at higher temperatures (Brahms & Mommaerts, 1964). The signals which appear before 220 nm in Fig 3.11, can be neglected due to impurities and contamination. The results in Fig 3.11 indicate that an Isodichroic or an Isbestic point appears around 270 nm to 281nm. Isodichroic point is the wavelength where the absorptivity is the same for two or more spectra of one sample(Corrêa & Ramos, 2009). The isodichroic points suggest a two-state melting process, which means the arrangement of the aromatic rings of the nucleases are different in the folded and melted state (Kypr & Vorlickova, 1986).

Fig 3.11 CD spectra of mRNA(Sirt1) at 25 °C, 50 °C, 75 °C and 82 °C.

To determine the wavelength with the best resolution for the mRNA(Sirt1), the differences in signal between the temperatures 25 °C and 82 °C was plotted at different wavelengths, Fig 3.12. To receive stronger signals and minimize the noise one can use a more concentrated sample.

Fig 3.12 The diagram shows the differences in signal between the temperatures 25 °C and 82 °C to determine the best wavelength for the melting studies. Here, it was clear that the highest difference in signal was around 268nm.

From the plot, Fig 3.12, the wavelength where the highest peak occured was chosen. The wavelength chosen was 268 nm. CD melting and annealing curves were recorded between the temperatures 17°C and 90 °C at 268 nm. The curves were fitted to the Hill’s equation described in the methods. The melting and folding curves are shown in Fig 3.13.

Fig 3.13 Ilustrates the CD folding and CD melting curves before and after fitting to the Hill’s equation.

To determine the melting temperature the values after the fitting are described in Fig 3.14. The melting temperature for mRNA(Sirt1) is around 44 °C.

Fit Tm Max Hill’s coefficient

Annealing 45.6 0.97 4.3 Melting 43 1 6

Fig 3.14 Showing the values after the CD curves were fitted to the Hill’s equation. It was possible to fit a sigmoidal curve to the anneling curve and it shows the melting temperature to be 45.6 °C. The melting curve can not be represented by a sigmoidal curve according to the Hill’s equation, because it looks more like a linear curve before fitting. The results show that the melting point for the mRNA(Sirt1) is between 43 °C and 45.6 °C, so it melts around 44 °C. At 44 °C the structure changes from an unordered to an ordered conformation in the annealing reaction. The Hill’s coefficient for the reaction is a measure of the cooperatitivty of the RNA folding (Rinnenthal et al., 2010). In both the CD melting the CD annealing curve low values of the Hill coefficient were received, which indicate that the transition between the folded and the unfolded RNA conformations happended gradually. This according to Rinnenthal et al. (2010).

How does the elucidation of mRNA(Sirt1) look after Circular Dichcroism study?

The results indicate, that maximal secondary structures appear around 260 nm and the amount of secondary structures decrease with elevated temperature. An Isodichroic point appears around 270 nm to 280 nm, which indicate a two state melting transition. The wavelength chosen for the temperature dependent analysis was 268 nm which gave the best resolution. To the CD melting and folding curves a sigmoidal line was fitted and the melting point was determined to 44°C. The low values for the Hill’s coefficient indicate that the transition between the two states happen gradually. The sample was very diluted so a more concentrated sample would have been preferred when analyzing with CD spectroscopy.

4. CONCLUSION

Here conclusions will be drawn from the results and discussion in accordance with the problem specification of this thesis.

The experiments from the optimization reaction indicate that the mRNA(Sirt1) sequence is highly ATP dependent and suggest that the ratio between ATP/CTP should be kept 1:2. Tris, Mg2+, PEG and GMP should also be added to increase the yield of mRNA(Sirt1). The polymerase concentration should also be considered, as in the case of this study less amount than expected was needed in order to maximize the product. The optimization was successful and we could continue to produce mRNA(Sirt1) sample for structural studies. mRNA(Sirt1) was purified with the final yield of 0.02%. The purified mRNA(Sirt1) still contained a weak plus one band, which was difficult to eliminate even after transcription reaction, precipitation and two purifications. Optimal yield was not obtained after the transcription reaction due to the many side products, which showed up on the gels during the optimization. The results indicate, that HPLC might be a better solution for purifying mRNA(Sirt1) instead of the method used in this thesis. The purification process contained different steps and a lot of mRNA(Sirt1) was lost during the process.

CD measurements was done on the purified mRNA(Sirt1) and with the help of the melting profiles it was clear that the secondary structures decreased with elevated temperature, indicating that the mRNA(Sirt1) is folded at 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.

5. FURTHER RESEARCH

Using CD spectroscopy melting profiles over the mRNA(Sirt1)-miR-34a complex can be done similarly to the ones done only for mRNA(Sirt1). In this way structural changes could be verified by peaks with probably higher intensity, due to miR-34a binding to mRNA(Sirt1).

It would also be interesting to purify the mRNA(Sirt1) with HPLC to receive higher yield and more pure mRNA(Sirt1). This method is under development it likely will improve yields tremendously. Then comparison with the melting curves obtained in this thesis could be done.

Further, I have optimized the mRNA(Sirt1) sample so that NMR studies can be carried out determining the structure of the mRNA(Sirt1)-miR-34a complex using labeled nucleotides in the large-scale transcription reaction.

6. UTBILDNINGSKAPITEL

Detta kapitel består av teori kring Samhällsfrågor med Naturvetenskapligt Innehåll (SNI-fall), forskning i klassrummet samt en beskrivning till det utbildningsmaterial som har utformats och testats för att avgöra hur det kan utgöra ett komplement i undervisningen av kemi 2 i gymnasieskolan.

6.1 Inledning

En diskussion som ständigt uppkommer är att skolresultaten försämras i Sverige i jämförelse med andra länder (Sjöberg, 2010). Forskare arbetar flitigt med att observera och analysera interaktionen mellan lärare och elever, för att beskriva elevernas inlärningssätt. Stor del av den didaktiska forskningen som görs drar slutsatser som visar på att viktiga parametrar i elevers inlärning är beroende av den sociala miljön, genom exempelvis dialoger och utbyte av erfarenheter (Björklid & Fischbein, 1996). I det naturvetenskapliga klassrummet saknas diskussioner kring frågor som berör hela samhället där eleverna kan utgå från sina egna erfarenheter för att få igång en diskussion. Av denna anledning skapades ett SNI-fall.

Det utbildningsmaterial som skapats under detta projekt ska fungera som ett komplement i kemiundervisningen på gymnasieskolor. Materialet är byggt som ett SNI-fall, vilket står för Samhällsfrågor med Naturvetenskapligt Innehåll. Det innebär att det i klassrummet arbetas med situationer som uppstår utanför skolan genom att använda elevernas egna erfarenheter. På så sätt blir eleverna mer intresserade samtidigt som de blir medvetna om viktiga samhällsfrågor (Ekborg m.fl., 2012).

För att inkorporera ny naturvetenskaplig forskning i klassrummet måste viktiga samhällsfrågor relaterat till den nya forskningen lyftas fram. En stor fråga som diskuteras i dagens samhälle är genmanipulation, där forskare har kommit så långt i utvecklingen av nya metoder att de med enkelhet kan substituera arvsmassan i grödor, växter och djur för förbättrade egenskaper hos dessa(Schylter, 2012). Genmanipulation har skapat stora möjligheter, som exempelvis att skapa mat till den allt mer växande befolkningen och möjligheten att bota svåra sjukdomar hos människor, men en intressekonflikt och en mängd etiska frågor har uppstått kring detta. Här ställs forskare mot människorättsorganisationer såsom Greenpeace som har olika åsikter och synpunkter om hur genmanipulation ska användas i samhället. Frågor kring var gränsen för genmanipulation går, vad som är rätt och fel men även vem som är ansvarig för hur genmanipulation får användas diskuteras flitigt.

Utbildningsmaterialet syftar till att ge eleverna förståelse för vad genmanipulation är, genom att skapa ett SNI-fall som grundar sig i en argumentationsuppgift där eleverna ska diskutera och argumentera denna svårsamhällsfråga utifrån egna erfarenheter och information som de tagit fram. Detta material har testats av en kemilärare och genom kvalitativa intervjuer med läraren och en enkätundersökning har materialet utvärderats utifrån de förmågor som eleverna kan träna med hjälp av SNI-fallet. En analys av elevernas argumentation har även gjorts för att utvärdera vilka typer av argument eleverna värdesätter kring frågan om genmanipulation.

6.2 Litteraturstudie

6.2.1 Forskning i klassrummet

Naturvetenskap upplevs av många som svårt i jämförelse med andra ämnen och detta är inte på grund av att intresset för ämnet inte existerar utan för att eleverna är kritiska till sättet det lärs ut. Eleverna känner att de inte vet varför de behöver den kunskapen, vilket har bidragit till att intresset minskat för ämnet (Ekborg m.fl., 2009).

Didaktisk forskning görs ständigt för att förbättra undervisningen för att bidra till bättre förståelse hos elever. I boken Forskningen för klassrummet, vetenskaplig grund och beväpnad erfarenhet i klassrummet som tagits fram av Skolverket beskriver vikten av att använda forskning för att utveckla befintliga mönster i pedagogiken samtidigt som eleverna får bättre utvecklingsmöjligheter(Minten, 2013). Pedagogiken som läraren tillämpar är av stor vikt och skillnaden mellan de olika typerna påverkar elevernas inlärning. Skillnaden på förmedlingspedagogik och lärarledd undervisning är stor. Förmedlingspedagogik innebär att läraren är den som talar mest, medan lärarledd undervisning handlar om att läraren skapar ett klimat i klassrummet där dialog och diskussion är tillåtet och på så vis främjar inlärningen hos elever(Minten, 2013). Detta kräver av läraren att denne har en tydlig ledarroll där eleverna känner sig trygga och vågar delta i dialoger och diskussioner där egna erfarenheter kan komma till uttryck. Detta sociokulturella perspektiv på lärande skapar en miljö som är gynnsam för elevers utveckling och lärande. Lev Vygotsky talar mycket om detta perspektiv(Vygotsky, 2007). Vygotsky grundade bland annat ett begrepp om teorier om lärande vid namn den proximala utvecklingszonen(ZPD) som är då eleven är i en situation som förgyller dess inlärning och utveckling. Denna består av tre delar som kan ses i Figur 1, en del är då individen klarar av uppgiften själv utan hjälp från läraren eller annan elev. Den andra delen är då eleven behöver hjälp av läraren eller annan elev för att lösas uppgiften då den är för svår för eleven att lösa på egen hand. Mellan dessa befinner sig den proximala utvecklingszonen som uppnås av individen då egna erfarenheter måste utnyttjas för att lära sig något nytt eller lösa ett problem, vilket sker i det sociala samspelet mellan exempelvis en lärare och en elev. Det är inom denna zon som barnet kan utvecklas(Björklid & Fischbein, 1996). Detta kräver att uppgiften som eleven får är på rätt nivå så eleven har möjlighet att utvecklas. Genom att läraren skapar möjligheter för eleverna att interagera och utbyta erfarenheter inom nyaområden och frågor kan elevernas intresse väckas samtidigt som de utvecklar en mängd olika förmågor.

Problemlösningen är utanför individens Självständigt arbete förmåga. Problemet kan med problemlösning ZPD inte lösas av individen även med hjälp.

Figur 1: Illustrering av Vygotskys begrepp den proximala utvecklingszonen(ZPD).

Vygotsky talar även om ett annat begrepp vid namn Child-in-Activity-in-Cultural-Context som har linkande infallsvinkel som ZPD men som syftar på att eleverna behöver förmedla och diskutera sin ”kultur”. Vygotsky menar att individen formas med hjälp av omgivningen, genom diskussioner, argument och motargument(Vygotsky, 2007).

6.2.2 Ämnesplaner skolverket

I ämnesplanen för ämnet Kemi 2 på gymnasiet beskrivs de förmågor och det centrala innehåll som eleven ska uppnå vid avklarad kurs. Här ingår att elever ska behandla etiska frågor genom att kommunicera kring hur det påverkar individ och samhälle utifrån den kunskap de lärt sig i kemiämnet(Skolverket, 2014). Nedan beskrivs några centrala innehåll i kursen kemi 2 och en förmåga som är direkt relaterade till detta arbete.

Centralt innehåll i Kemi 2: • Det genetiska informationsflödet, inklusive huvuddragen i de biokemiska processerna replikation, transkription och translation. • Frågor om etik och hållbar utveckling kopplade till kemins olika arbetssätt och verksamhetsområden.

En förmåga som ska uppnås i Kemi2: • Kunskaper om kemins betydelse för individ och samhälle.

Ett av kemiämnets syften beskriver vikten av att elever ska få möjlighet att skapa sig ett naturvetenskapligt perspektiv på samhället. Detta ska göras genom att elevernas egna erfarenheter och nyfikenhet tas till vara genom diskussion av aktuell forskning.

“Undervisningen ska också bidra till att eleverna, från naturvetenskaplig utgångspunkt, kan delta i samhällsdebatten och diskutera etiska frågor och ställningstaganden.”(Skolverket, 2014)

Här poängteras vikten av att eleverna i dialog ska få fördjupa sina kunskaper genom att ta del av samhällsfrågor och aktivt få diskutera svåra etiska frågor. Douglas Robert tog fram begreppet emfaser som beskriver vad undervisningen förmedlar till eleverna, både utifrån undervisningsstil men även utifrån material såsom läromedel. Det finns sju emfaser, men den som bäst kan relateras till syftet för ämnet Kemi 2 är Vetenskap, normer och beslut. Denna emfas innefattar att en elev ska kunna diskutera samhällsfrågor utifrån ett 46 naturvetenskapligt perspektiv. I denna emfas är värderingen i beslutsprocesser viktig så eleven kan skilja på naturvetenskap och värderingar(Roberts, 1982). För att eleverna ska känna at naturvetenskapen är viktig och att de har nytta av det utanför skolan måste nya idéer formuleras vad gäller formning av undervisningen. Forskning har visat att elevernas intresse väcks då de får arbeta med ett riktigt problem där de får tillämpa sina naturvetenskapliga kunskaper (Ekborg m.fl. 2009). SNI-fall, är ett arbetssätt där läraren kan skapa material utifrån samhällsfrågor som kan bearbetas av eleverna genom diskussioner och använda sina naturvetenskapliga kunskaper relaterat till ämnesplanen.

6.2.5 Samhällsfrågor med naturvetenskapligt innehåll SNI-fall är ett sätt att få elever att engagera sig i frågor som rör samhället genom att eleverna är delaktiga och utnyttjar sina egna åsikter och idéer för att skapa lärande (Ekborg m.fl. 2012). Genom att knyta elevernas tidigare erfarenheter till att diskutera samhällsfrågor har läraren verklighetsanpassat undervisningen samtidigt som elevernas intresse väckts. Då eleverna får ta sig an och diskutera relevanta samhällsfrågor lär de sig att ta ställning på ett källkritiskt sätt (Ekborg m.fl. 2012).

”Den ska leda till att eleverna utvecklar förståelse av hur naturvetenskapliga kunskaper kan användas i såväl yrkesliv som vardagsnära situationer och för att göra personliga val och ställningstaganden.”(Ekborg m.fl. 2012, s.22)

Elverna ska arbeta med dessa SNI-fall i grupp för att främja deras lärande, genom diskussioner och argumentationer kring tidigare erfarenheter. Forskning har visat att det mänskliga lärande är en social process som sker genom interaktion mellan elever och lärare(Björklid & Fischbein, 1996). Ytterligare forskning har visat att elever måste få arbete med uppgifter såsom SNI-fall för att kunna utveckla förmågorna att analysera och förstå situationer för att sedan kunna ta ställning i frågor(Ekborg m.fl. 2009). Vilket även är det sociokulturella perspektiv som bland annat Vygotsky talar om kring lärande(Vygotsky, 2007).

6.3 Syfte och frågeställning

Syftet med detta arbeta är att skapa ett utbildningsmaterial som är utformat som ett SNI- fall som väcker elevers intresse kring frågor som berör hela samhället relaterat till ämnesplanen i kemi 2. Till skillnad från Ekborg m.fl.(2012) som hanterar SNI-fall i grundskolan genomförs denna studie i gymnasieskolan med vikt på ny naturvetenskaplig forskning samt etiska frågor som uppkommer.. Det ämne som har valts till SNI-fallet är genmanipulation och de etiska frågor som dyker upp kring ämnet. Materialet som skapats kommer att testas av en lärare på en grupp elever för att analyseras och utvärderas. Utvärderingen av materialet kommer ske med avseende på de förmågor som kan tränas med hjälp av SNI-fallet genom enkäter och intervjuer. De olika typer av argument som eleverna väljer för att ta ställning i de etiska frågorna kommer att analyseras och kategoriseras.

- Vilka förmågor tränas med hjälp av utbildningsmaterialet det skapade utbildningsmaterialet utifrån några gymnasieelever och en gymnasielärares perspektiv? - Vilka argument använder elever i diskussion om genmanipulation med hjälp av utbildningsmaterialet?

6.4 Metod

Det utbildningsmaterial som skapats är ett SNI-fall som berör ämnet genmanipulation, bilaga 1. Ämnet har valts ut på grund av dess direkta koppling till ämnesplanen i Kemi 2 och kan relateras till framtida forskning. Detta material kan användas som ett komplement i kemiundervisningen. Materialet har testats av en lärare på två olika elevgrupper vardera bestående av 8 elever. För att svara på den första frågeställningen fick eleverna besvara enkäter och en kvalitativ intervju med läraren genomfördes. Elevernas nedskrivna argument samlades in och analyserade för att besvara den andra frågeställningen för detta arbete.

6.4.1 Datainsamling

För att kunna svara på frågan om vilka typer av argument elever använder för att diskutera de etiska frågorna kring genmanipulation valdes att samla in elevernas skriftliga argument som de förberett inför lektionen. Denna datainsamling var av kvantitativt värde och argumenten som eleverna valde kunde sedan kategoriseras enligt Sjöbergs olika argumentationstyper(Sjöberg, 2010). Kategorierna för dessa argumentations typer är Demokratiargument, Ekonomiargument, Nyttoargument och Kulturargument. Utifrån argumenten skapades ett diagram där trenden i de argumentationer eleverna använder kunde diskuteras och analyseras.

För att kunna få en djupare inblick i vilka förmågor eleverna kan träna med hjälp av utbildningsmaterialet gjordes en enkätundersökning som eleverna fick delta i, som genomfördes enligt vetenskapsrådets grunder. Enkäten utformades i form av en alternativfråga, där eleverna fick välja ett eller flera alternativ, om de olika förmågorna som materialet speglar(Bell, 2006). Detta samlades in i ett dokument där ett diagram utformades.

En kvalitativ intervju, bilaga 3, genomfördes med läraren som testade materialet för att få en bild över vilka förmågor läraren tyckte kunde tränas med materialet. Intervjun varade i 30 minuter. Fördelen med att genomföra kvalitativa intervjuer är att informationen som framgår är mycket mer än bara ett svar på en fråga, utan den som intervjuar kan ur den respons som fås få ut mer information genom att studera tonfall, mimik och pauser(Bell, 2006). Läraren som blev intervjuad fick långt innan intervjun information om syftet med intervjun samt vad informationen som kommer fram under intervjuns gång kommer att användas till. Frågorna som skulle ställas under intervjun skickades till läraren som skulle testa materialet för att göra intervjun så tydlig som möjlig(Bell, 2006). Intervjun med läraren var utformat som en semistrukturerad intervju, där ett antal frågor och uppföljningsfrågor angående de olika förmågorna som materialet innefattar förbereddes. Valet av intervjumetod gjordes så läraren kan känna en frihet till att ta upp saker som för hen anses vara viktiga samtidigt som det finns en viss struktur(Bell, 2006). Intervjun skrevs ner med hjälp av papper och penna för att ge en mer personlig känsla gentemot läraren. Förmågorna som läraren beskriver jämfördes sedan med de förmågor som eleverna ansåg materialet speglade.

6.4.2 Urval och kvalitetsredovisning

Undersökningen genomfördes på två grupper om 8 elever där eleverna delades in i grupper om 4 elever. Elverna var från två olika gymnasieskolor i Sverige och dessa elever gick åk 3 och var 18 år. Dessa elever informerades i god tid om undersökningen och hade möjlighet att avstå i fall de inte ville delta. Studien genomfördes enligt vetenskapsrådets forskningsetiska principer(Vetenskapsrådet, 2002). Totalt 16 elever deltog i undersökningen

48 där de hade förberett uppgiften hemma och genomförde en diskussion under 1 timme i klassrummet. Efter lektionen lämnade de in sina argument som de förberett samtidigt som de besvarade en enkätundersökning. Intervjunundersökningen som genomfördes med en lärare var av hög validitet då intervjun genomfördes som en semistrukturerad intervju gav det mycket mer information än förväntat. När det gäller intervjun, enkätundersökningen samt de insamlade argumenten har dessa hög validitet men eftersom det endast var en liten grupp som deltog i undersökningen är generaliserbarheten liten. Resultaten av denna undersökning representerar endast en liten del av Sveriges elever och lärare i avseende till detta material.

6.5 Resultat

Enkätundersökningen, den kvalitativa intervjun och de insamlade argumenten har sammanställts och beskrivs nedan var för sig med hjälp av diagram.

6.5.1 Förmågor som tränas med hjälp av SNI-fallet utifrån elevperspektiv

Resultaten från enkätundersökningen som eleverna var med och besvarade efter att de arbetat med det skapade materialet visas i Figur 2. Elverna skulle kryssa i vilka förmågor de ansåg att de kunde träna men utbildningsmaterialet

Vilka förmågor tränar SNI-fallet ur elevers perspektiv 1! 2! 3! 4! 5!

10%!

30%! 20%!

40%!

Figur 2: Resultat över de förmågor eleverna anser de kan träna med hjälp av SNI-fallet de testat. 1. Kunskaper om kemins begrepp, modeller, teorier och arbetsmetoder samt förståelse av hur dessa utvecklas. 2. Förmåga att analysera och söka svar på ämnesrelaterade frågor samt identifiera, formulera och lösa problem. Förmåga att reflektera över och värdera valda strategier, metoder och resultat. 3. Förmåga att planera, genomföra, tolka och redovisa experiment och observationer samt förmåga att hantera kemikalier och utrustning 4. Kunskaper om kemins betydelse för individ och samhälle. 5. Förmåga att använda kunskaper i kemi för att kommunicera samt för att granska och använda information.

Utifrån diagrammet är det tydligt att 40 % av svaren som eleverna gav anser att SNI-fallet speglar förmågan, kunskaper om kemins betydelse för individ och samhälle. Sedan visade även svaren att 30 % anser att uppgiften speglar, förmåga att använda kunskaper i kemi för att kommunicera samt för att granska och använda information. Därefter kom förmågan att analysera och söka svar på ämnesrelaterade frågor samt identifiera, formulera och lösa problem. Förmåga att reflektera över och värdera valda strategier, metoder och resultat med 20 % och sedan Kunskaper om kemins begrepp, modeller, teorier och arbetsmetoder samt förståelse av hur dessa utvecklas med 10 %. När man tittar på diagrammet ser man att eleverna inte tycker att, förmåga att planera, genomföra, tolka och redovisa experiment och observationer inte kunde tränas med uppgiften de genomfört.

6.5.2 Förmågor som tränas med hjälp av SNI-fallet utifrån lärarperspektiv

Mycket värdefull information kunde uthämtas ifrån den semistrukturerade intervju som genomfördes med den lärare som testade materialet. Tyngdpunkten i samtalet handlade om de förmågor som eleverna har möjlighet att träna med hjälp av SNI-fallet. Läraren beskrev att alla förmågor kunde tränas förutom förmåga 3, förmågan att planera, genomföra och tolka och redovisa experiment och observationer. Läraren berättade även att denna typ av SNI-fall bidrar till att eleverna utnyttjar sin naturvetenskapliga kunskaper såsom begrepp och termer för att genomföra uppgiften. För att kunna stödja sina argument måste eleverna söka information och avgöra hur de kan koppla det till individ och samhälle. Nedan visas ett citat från intervjun med läraren där läraren beskriver ett intryck från eleverna under genomförandet av uppgiften.

”Eleverna var positiva och tyckte det var en intressant uppgift och tycker det är viktiga och svåra uppgifter att ta ställning till”- Läraren som intervjuades

6.5.3 Argument som elever använder i diskussion om frågan genmanipulation med hjälp av SNI-fallet

Argumentationerna som eleverna lämnade in efter att de genomfört debatten i klassen analyserades och kategoriserades utifrån Sjöbergs fyra argument för de naturvetenskapliga ämnena i skolan. Dessa fyra kategorier är Ekonomiargumentet, Nyttoargumentet, Demokratiargumentet och Kulturargumentet(Sjöberg, 2010). Nedan i figur 3 visas ett diagram över de olika argumentationstyper eleverna valde att använda.

Kategorisering enligt Sjöbergs fyra argumentations typer Ekonomiargument! Nyttoargument! Demokratiargument! Kulturargument! 25%! 38%!

25%! 13%!

Figur 3: Visualiserar de olika typer av argument som eleverna tog upp i arbetet med SNI-fallet. 50

En del typer av argument som eleverna tog upp i sina uppgifter representeras nedan för att ge en inblick över hur kategoriseringen skett.

Demokratiargument:

Sjöberg(2010) beskriver att i en demokrati har människor rätt att påverka sin egen situation. För att kunna vara en demokrati krävs det att att befolkningen har kunskap och tillräckligt god kompetens i naturvetenskapliga ämnen. Nedan visas ett citat ur en elevs argumentations uppgift som enligt Sjöberg kan kategoriseras som ett Demokratiargument.

”En omröstning där frågan om genmanipulation ska få genomföras eller inte bör göras så ett gemensamt beslut kan tas.” - Elev X

Eleven vill visa att genmanipulation är en fråga för alla som måste beslutas tillsammans. I flera elevers argument framkom även svårigheten att låta det bli ett majoritetsbeslut och att det kan leda till att de förbättrade egenskapen kommer skapa en elit och att alla mer eller mindre blir tvungna till att genomföra det för att kunna ha en chans mot alla andra. På samma sätt följer för resterande kategorier.

Ekonomiargument:

”Näringslivet är för en progressiv utveckling av genmanipulation eftersom man kan få fram livsmedel som ser bättre ut, smakar bättre och man kan producera i stora volymer, väldigt billigt och effektivt för olika företag som på så vis gynnas”- Elev X

Ekonomiargumenten som eleverna tog upp kretsade mycket kring att genmodifierad mat och grödor är ekonomiskt gynnsamt. Eleverna påpekar att pågrund av de dåliga egenskaperna hos mat och grödor exkluderas blir de billigare och att produktionen kan öka.

Nyttoargument:

”Genmanipulation kan hjälpa till med svälten då genmanipulation kan öka skördar genom större grödor som samtidigt är mindre känsliga. Då finns det gott om mat för alla och det blir billigare.” - Elev X

Denna argumentations kategori använder eleverna sig av för att visa vad som faktiskt kan genomföras med hjälp av genmanipulation och att det kan bidra till att minska svälten på jorden. De diskuterar samtidigt om hur det kommer att bli om alla människor får leva som de gör i Sverige och att det kan ha negativa konsekvenser för jorden.

Kulturargument:

”Vill inte att det ska sluta med att man förbjuder vissa gener på grund av sjukdom eller på grund av till exempel en diktator. Dock kan det vara bra att kunna ändra om generna om barnet riskerar att dö eller liknande.”- Elev X

Kulturargument var de typer av argument som framgick mest i elevernas argumentationsuppgifter. Här tog eleverna fram argument som vad som skulle hända ifall gentekniken hamnar i fel händer och hur det då kan påverka människan och samhället. Eleverna tog flitigt fram argument om hur rättigheterna att genomföra tester på djur ser ut och påpekade att djuren också har rättigheter.

Ur diagrammet, Figur 3, som illustrerar de olika kategorier av argument som eleverna valde att ta upp. Viktigt att notera är att varje elev skrev ned mer än ett argument vilket bidrar till att diagrammet är mer av en representation av vilka typer av argument eleverna helst tillämpar under diskussionen av detta SNI-fall. Det är tydligt att Kulturargument är det mest förekommande bland eleverna då de vill ta ställning i frågan om genmanipulation, men även Ekonomiargument och Nyttoargument tillämpas flitigt. Demokratiargument har färre använt sig av visar diagrammet.

6.6 Analys & Diskussion

Utifrån enkätundersökningen och den kvalitativa intervjun kunde den första frågan som behandlar de olika förmågorna som kan tränas med hjälp av det skapade SNI-fallet besvaras. Det visade sig att de förmågor som läraren anser eleverna kan träna stämmer överens med det som eleverna tycker att de har möjlighet att öva med hjälp av materialet. Enligt enkätundersökningen ansåg eleverna att den förmåga som de främst kunde träna var, kemins betydelse för individ och samhälle vilket stämmer överens med syftet av SNI- fallet. Eleverna var positiva till uppgiften och den bjöd in till argumentation där de kunde använda sig av sina egna erfarenheter samtidigt som de kunde använda sina naturvetenskapliga kunskaper för att sätta sig in i frågan som SNI-fallet byggde på. Den förmåga som både eleverna och läraren inte alls tyckte de kunde träna var förmågan att planera, genomföra, tolka och redovisa experiment och observationer samt hantera kemikalier och utrustning. På det sätt som SNI-fallet är utformat finns inget utrymme för att uppvisa denna förmåga, men som det framgick i intervjun med läraren finns möjligheter att utveckla materialet vidare genom exempelvis studiebesök eller liknande där denna förmåga också kan inkorporeras. Målet med det skapade SNI-fallet var att låta eleverna använda sina naturvetenskapliga kunskaper för att jobba med den svåra frågan som berör genmanipulation och kunna ta ställning. Genom att eleverna anser att de har möjlighet att träna de flesta förmågorna med denna typ av uppgift, visar vikten av att arbeta på ett sociokulturellt sätt. Där ett lärarlett moment som denna kan bidra till en miljö där eleverna har utrymme att växa och utvecklas på samma gång som de uppnår de förmågor och det centrala innehåll som beskrivs i ämnesplanen för Kemi 2. Som beskrivits tidigare saknas det dialog och diskussion i det naturvetenskapliga klassrummet och SNI-fall är ett sätt att tillämpa dessa kunskaper anpassat till frågor som uppkommer i det vanliga livet utanför skolan. På detta sätt lär sig elever skilja på naturvetenskap och värderingar genom uppgifter som tangerar en av Roberts emfaser(1982) som är Vetenskap, normer och beslut. Detta SNI-fall tränar eleverna på att ta sig an en viktig samhällsfråga där de måste planera, söka information och på ett naturvetenskapligt sätt skapa argument och motargument för att diskutera de etiska frågorna som frågan berör genmanipulation både ur ett individperspektiv och ur ett samhällsperspektiv.

Ur kategoriseringen som gjordes med hjälp av Sjöbergs fyra argument är det en tydlig trend som visar att eleverna helst använder sig av Kulturargumentet som existerade kring detta SNI-fall. Det argument som eleverna använde sig minst av var Demokratiargumentet och i de få fall de valde att använda sig av det kom de fram till att frågan om genmanipulation är en för svår fråga att behandla genom en enkel folkomröstning. Främst diskuterade eleverna fallet då genmanipulation skulle komma att genomföras på människor för att ge bättre egenskaper och minska sjukdomar, då menade eleverna att alla automatiskt kommer bli indragna och bli tvungna till att genomföra det trots att de kanske inte vill men att de inte har något annat val, eftersom de kommer hamna efter om de är ”normala” och inte förändrar något. Av denna anledning avstod eleverna från att använda sig av demokratiargument på grund av de svårigheter som existerar kring frågan om genmanipulation. 52

6.7 Slutsats

Slutsatsen i denna studie är att de flesta förmågor som finns med i ämnesplanen för Kemi 2 kan tränas med hjälp av det skapade SNI-fallet där eleverna genom diskussion och argumentation kan utnyttja sina kunskaper i naturvetenskap till att ta ställning i svåra samhällsfrågor. De argument som eleverna helst använde för att ta ställning i frågan om genmanipulation som SNI-fallet berör är Kulturargument.

6.8 Vidare forskning

Denna undersökning som har gjorts visar på vad en liten grupp elever tycker om denna typ av SNI-fall. Det representerar de förmågor eleverna och en lärare anser uppgiften tränar. En intressant härledning skulle kunna vara att genomföra undersökningen med fler elever och lärare för att få en djupare inblick över de olika förmågorna som kan tränas med SNI- fall. Det skulle även vara intressant att genomföra observationer över de olika typer av argument och begrepp som eleverna använder då de diskuterar uppgiften, vilket inte var möjligt att göra under denna undersökning på grund av tidsbrist.

7. REFERENCE

7.1 Articles and books

Scientific references Atkins, P & Paula, J. (2010). Physical Chemistry. 9th edition. Oxford.

Brahms, J & Mommaerts, W. (1964). A Study of Conformation of Nucleic Acids in Solution by Means of Circular Cichroism. J. Mol. Biol. pp.73-88

Beckert, B & Masquida, B. (2011). Synthesis of RNA by In Vitro Transcription.. RNA, Methods in Molecular biology. Vol 703. pp. 29-41.

Campbell, M. & Farrell, S. (2012). Biochemistry. 7th edition, International Edition. ChembioChem

Cloonan, N. (2015). Re-thinking miRNA-mRNA interactions: Interwining issues confound target discovery. BioEssays. Vol 37. pp. 379-388.

Corrêa, D & Ramos, C. (2009). The use of circular dichroism spectroscopy to study protein folding, form and function. African Journal of Biochemistry Research. Vol 3. pp. 164-173.

Fujita, Y. Kojima, K. Hamada, N. Ohhashi, R. Akao, Y. Nozawa, Y. Deguchi, T & Ito, M.(2008). Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells. Biochemical and Biophysical Research Communications. Vol 377. pp. 114-119.

Jasinski, L. Schwartz, C. Haque, F & Guo, P. (2015). Large Scale Purification of RNA Nanoparticles by Preparative Ultracentrifugation. RNA Nanotechnology and Therapeutics Methods in Molecular Biology. Vol 1297. pp. 67-82.

Kypr, J & Vorlickova, M. (1986). Graphical analysis of Circular Dichroic Spectra Distinguishes between Two-State and Gradual Alterations in DNA Conformation. Gen. Physiol. Biophys. pp. 415-422

Köhrer, K & Domdey, H. (1991). Preparation of High Molecular Weight RNA. Methods in Enzymology. Vol 194. pp. 398-405.

Lee, J. Padhye, A. Sharma, A. Song, G. Miao, J. Mo. Wang, L & Kemper, JK. (2010). A Pathway Involving Farnesoid X Receptor and small Heterodimer Partner Positively Regulates Hepatic Sirtuin 1 levels via MicroRNA-34a Inhibition. The Journal of Biological Chemistry. Vol 285. pp. 12604- 12611.

LePecq, JB & Paoletti, C. (1967). A Fluorescent Complex between Ethidium Bromide and Nucleic Acids Physical-Chemical Characterization. Journal of Molecular Biology. Vol 27. pp. 87-106.

Loening, UE. (1967). The Fractionation of High-Molecular-Weight Ribonucleic Acid by Polyacrylamide-Gel Electrophoresis. Department of Botany, University of Edinburgh.

Major. F & Parsien.M. (2008). The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature. Vol 452. pp. 51-55. 54

Misso, G. Di Martino, M. De Rosa, G. Farooqi, A. Lombardi, A. Campani, V. Zarone, M. Gullá, A. Tagliaferri, P. Tassone, P & Caraglia, M.(2014). Mir-34: A New Weapon Against Cancer?. Molecular Therapy-Nucleic Acids.

Pasquinelli, AE. (2012). MicroRNAs and their targets: recognition, regulation an emerging reciprocal relationship. Nature Reviews Genetics13. pp. 271-282

Petrov, A. Wu, T. Puglisi, E & Puglisi, J. (2013). RNA Purification by preparative Polyacrylamide Gel Electrophoresis. Methods in Enzymology Vol 530. pp. 315-330

Pugsli, J & Tinoco, I. (1989). Absorbance MElting Curve of RNA. MEthods in enzymology. Vol 180. pp. 304-325.

Rinnental, J. Klinkert, B. Naberhaus, F and Schwalbe, H. (2010). Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution. Vol. 38. pp. 3834-3847. Nucleic Acid Research.

Rokavec, M. Li, H. Jiang, L & Hermeking, H. (2014). The p53/miR-34 axis in development and disease. Journal of Molecular Cell Biology. Vol 0, pp. 1-17.

Schenborn, E & Mierendorf, R. (1985). A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acid Research. Vol.13.pp. 6223-6136.

Sharma, P. Mitra, A. Sharma, S & Singh, H. (2007). Base pairing in RNA structures: A computational analysis of structural aspects and interaction energies. Vol 119. pp. 525-531. Center for Conputational Natural Sciences and Bioinformatics.

Sosnick, T. Fang, X & Shelton, V. (2000). Application of Circular Dichroism to Study RNA Folding Transitions. Methods in enzymology. Vol 317. pp. 393- 409.

Yamakuchi, M. Ferlito, M & Lowenstein, C. (2009). MiR-34, SIRT1 and p53. The feedback loop. Departements of Medicine and Pathology; The Johns Hopkins University School of Medicine; Baltimore, Maryland, USA.

Weissman, D. Pardi, N. Muramatsu, H & Karikó, K. (2013). HPLC Purification of In Vitro Transcribed Long RNA. Syntetic Messenger RNA and Cell Metabolism Modulation Methods in Molecular Biology. Vol 969. pp. 43-54.

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Pedagogical references Bell, J. (2006). Introduktion till forskningsmetodik. Fjärde upplagan.

Björklid, P. Fischbein, S. (1996). Det pedagogiska samspelet. Studentlitteratur, Lund.

Ekborg. Ideland. Lindahl. Malmberg & Ottander & Rosberg. (2012). Samhällsfrågor i det naturvetenskapliga klassrummet. Första upplagan. Gleerups Utbildning AB.

Ekborg. Ideland & Malmberg. (2009). Science for life-a conceptual framework for construction and analysis of socio-scientific cases. NORDINA.

Hägg. Kerstin & Kuoppa. Svea Maria. (2007). Professionell vägledning – med samtal som redskap. Lund: Studentlitteratur.

Kullberg, B. (2004). Etnografi i klassrummet. Upplaga 2:8. Studentlitteratur AB, Lund.

Lundahl, C. (2012). Bedömning för lärande. Första upplagan. Norstedts.

Minten, E. Skolverket. (2013). Forskning för klassrummet vetenskaplig grund och beprövad erfarenhet i praktiken.

Roberts, D.A.(1982). Developing the concept of curriculum emphases in science education.

Vetenskapsrådet. (2002). Forskningsetiska principer inom humanistisk-samhällsvetenskaplig forskning. Stockholm: Vetenskapsrådet.

Vygotskij, S. L. (2007). Tänkande och språk. Uddevalla: Bokförlaget Diadolos AB.

Ämnesplan i kursen Kemi 2. Hämtat från skolverkets webbsida 24.02.15: .http://www.skolverket.se/laroplaner-amnen-och- kurser/gymnasieutbildning/gymnasieskola/kem?tos=gy&subjectCode=KEM&lang=sv&courseCode= KEMKEM02#anchor_KEMKEM02

Artikel av Schlyter 2012 hämtat 26.04.15: http://www.svd.se/opinion/brannpunkt/genmanipulation-ger-inte-ett-uthalligt-jordbruk_6951821.svd

7.4 Graphics

Fig 1.1: Wikimedia Commons. (2015). Eukaryotic cell. [Picture] http://commons.wikimedia.org/wiki/File:Eukaryotic_Cell_(animal).jpg

Fig 1.6: Schmoop. (2015). Difference between DNA and RNA.[Picture] http://www.shmoop.com/dna/genetic-code.html

Fig 1.7:Limbic lab. (2015). Transcription reaction. [Picture] https://limbiclab.files.wordpress.com/2012/12/limbic_lab_dna_transcription_diagram1.p ng.

8. APPENDIX A - Lab protocol

Protocol for RNA preparation

1. Annealing Reaction(AR) Optimization a. Dilute each DNA strand to a final concentration of 100µM with water (#nmol*10 =µl of water to add)

b. Annealing reaction mix:

7,50 µl of reverse strand DNA 7,50 µl of T7 DNA primer 6,00 µl of ddH20 9,00 µl of MgCl2(0,01M) 30,00 µl reaction in total

c. Incubate the annealing reaction at 95°C for 5 minutes.

d. Put the annealing reaction on ice for 30 minutes, afterward the tubes can be stored in the freezer until needed. RNA degrades in room temperature!

2. Transcription Optimization (OT) Before doing the large-scale transcription reaction(5ml), small transcription reactions(50µl) are done in order to choose the best condition to receive the mot RNA yield as possible.

a. Preparing optimization samples. Start with the example transcription reaction which is in the excel file.

1. Mix all of the reactants in one tube for each setup 2. Preheat the samples for 5 minutes at 37°C. 3. Add the polymerase to the samples and mix carefully with the tip of the pipett. Take the polymerase out of the freezer ONLY when it will be used and try to keep it in the cool box as much as possible! 4. Incubate the transcription reaction samples at 37°C for 2 hours.

b. Preparing the gel solution and loading the samples in the polyacrylamide gel.

1. Clean and setup the gel plates 2. Preparation of the gel solution and stir vigorously after adding the ingredients i. 50ml of 20% polyacrylamide/8M Urea/1xTBE buffer ii. 300µl of 10% APS iii. 30µl of TEMED 3. Load gel solution into the plates and add the comb. Let gel polymerize 20 minutes. 4. Set up the gel plates into the gel box and fill the apparatus with TBE buffer and slowly remove the comb. Make sure the the gel plates are not to tightened because it can cause the glas to break!

5. Clean the wells with a syring containing TBE buffer to remove urea. 6. Preheat the gel for 30 minutes at 12W. 7. Prepare the sample 1µl of transcription sample 9 µl of RNA loading buffer 8. Clean the wells with buffer again and load the samples. Run the gel for 1,5 hours at 12W. Monitor the gel from time to time so the glas is not broken or the buffer leaking!

c. Analyze the gel

1. Take the top plate off carefully 2. Place the gel in the Ethidium Bromide for 10 minutes. Wear double gloves because Ethidium Bromide is dangerous and carcinogen! 3. Wash the gel in ddH20 for 5 minutes 4. Take a picture on UV-plate and determine the best optimization conditions.

3. Large-scale transcription(LT) a. Calculate the number of 30µl annealing reaction needed to obtain the amount of DNA needed for the large-scale transcription 5ml. Prepare the annealing reaction as described in 1, but prepare the amount of samples needed for LT.

b. Calculate the amount of each reactant needed for the LT reaction. Multiply each reactant with 100 and adjust the amount of water so the total volume of the sample is 5ml.

c. Mix all the reactants in a 50 ml falcon tube, incubate at 37°C for 5 minutes. d. Add 60µl of RNAse inhibitor e. Add polymerase and careful pipett. f. Incubate the transcription reaction at 37°C for 4 hours.

4. RNA purification a. Centrifuge the sample at 4900 rmp at 4°C for 30 minutes.

b. Use 0.2µm Millipore tubes to filter: 1. Connect Falcon tube with sample to filter, filter first time. 2.Add 1ml ddH20 to the pellet(phosphates). Vortex briefly if the pellet doesn’t dissolve. Filter this solution. Some samples don’t have any precipitate, just filter those once! 3. Clean the empty falcon tube with 1-3ml ddH20 and filter. 4. Concentrate the sample in the centrifuge at 4°C at 4900 rmp to 1ml with a clean amicon filter. The normal of the amicon filters themselves should be perpendicular to the rotation axis of the centrifuge so that the solution is pushed out through the filters. This takes about 20 minutes and do not let the filter run dry!

c. Prepare the large-scale 20% polyacrylamide/8M Urea/1XTBE 1. Prepare the amounts of the gel solution.

100ml of 20% polyacrylamide/8M Urea/1TBE 600µl of 10% APS 60µL OF TEMED 2. Add the gel solution to the plates and add the comb(with plastic wrap around it to create on large well). Let it polymerize for 45 minutes. 3. Remove the comb and add running buffer 4. Preheat the gel at 18W for 1 hour.

d. Mix the concentrated sample with 1 ml RNA loading buffer and heat it up for 5 minutes at 95°C. Do this right before you add it to the gel!

e. Load the sample to the gel and let it run for 3 hours at 18W. Do not runt it for too long, because you may loose RNA and monitor the position of the RNA!

f. Prepare the elution buffer: 1. Final volume of 250 ml 2. Make 10% sodium dodecyl sulfate(SDS) 3. Mix 10g of SDS with 100ml ddH20 and steri-filter the solution. 4. Make elution buffer by mixing 222,5ml of 10mM Tris-HCl,1mM EDTA (pH=8.0) with 25ml of 5M NaCl and 2.5ml of 10% SDS. 5. Steri-filter the solution

g. Take the gel down, remove the glas plates, and place the gel on top of a UV paper. Look at the bands with the UV light and find the band that corresponds to your RNA. Cut out the band in pieces and put the pieces through a syringe. Crush the pieces into a beaker and add 15 ml of elution buffer to it. Put plastic on top of the beaker and place it in the fridge overnight.

h. Collect the supernatant from the debris in an falcon tube and add another 10 ml elution buffer to the beaker with the debris and let it in the fridge for 2 hours. Repeat once again. Concentrate the collected supernatant with an amnion filter at 4900 rmp 4°C ut the total volume of RNA is 300µl.

5. Precipitation of RNA a. Put the RNA sample into a falcon tube. b. Add 10% volume of NaAc(3M, pH=5.2) and 3 times the volume of sample of 100% Ethanol. c. Vortex the solution and store in the freezer(-20°C) overnight. d. Prepare 70% EtOH solution and store in the freezer. e. Place the falcon tube with the sample in the centrifuge and spin down the sample for 2 hours at 4900 rmp at 4°C. Check for ice! f. Pipett the supernatant out of the tube. Your RNA should be precipitated at the bottom of the tube. Be careful so you don’t loose the RNA! g. Wash the pellet with ice cold 70% EtOH with the same volume as the previous solution. Do not shake! h. Spin down the sample for another 2hours at 4900 rmp at 4°C. i. Pipette the supernatant and store the tube in the freezer and cover with parafilm with holes in it so any left ethanol will evaporate.

6. Washing the RNA a. Wash the amicon filter with ddH20 and spin it down for 15 minutes.

b. Meanwhile, dissolve the RNA pellet with 1 ml ddH20 and raise the temperatur to 95°C for 5 minutes. Then cool it down in ice for 30 minutes. c. Add the sample to the amicon filter and spin the sample at 4900rmp at 4°C until 300µl of sample is left. d. Add 2ml of NMR buffer to the RNA sample and spin for 25 minutes. e. Repeat d twice and spin down until you have 250µl of NMR buffer and sample left. f. Store the sample in the freezer and calculate the concentration of the sample. https://eu.idtdna.com/calc/analyzer (The extinction coefficient can be found in the link)

9. APPENDIX B - Calculations

Calculations after the first purification of mRNA(Sirt1)

The measured absorbance obtained with Nano Drop for each sample was written down and the extinction coefficient found for mRNA(Sirt1). Using Lambert-Beers law the concentration was calculated.

�!"# �!"#$ !"#$! = �!"#$ !"#$! ∗ �

SAMPLE 1

�!"# = 0.256

� � = 263300 !"#$(!"#$!) (�� ∗ ��) � = 1 ��

�!"# 0.256 !! �! = = = 9.7 ∗ 10 � �!"#$ !"#$! ∗ � 263300 ∗ 1

SAMPLE 2

�!"# = 0.860 � � = 263300 !"#$(!"#$!) (�� ∗ ��) � = 1 ��

�!"# 0.860 !! �! = = = 3.27 ∗ 10 � �!"#$ !"#$! ∗ � 263300 ∗ 1

SAMPLE 3

�!"# = 0.144 � � = 263300 !"#$(!"#$!) (�� ∗ ��) � = 1 ��

�!"# 0.144 !! �! = = = 5.5 ∗ 10 � �!"#$ !"#$! ∗ � 263300 ∗ 1

The three samples were put together and the total mass of the product was calculated.

!! !! !! !! �!"!#$ = 9.7 ∗ 10 + 3.27 ∗ 10 + 5.5 ∗ 10 = 4.79 ∗ 10 �

!! !! !! �!"#$ !"#$! = �!"!#$ ∗ �!"!#$ = 4.79 ∗ 10 ∗ 3 ∗ 10 = 14.37 ∗ 10 ��

!! !! �!"#$ !"#$! = �!"#$(!"#$!) ∗ �!"#$ !"#$! = 8471.5 ∗ 14.37 ∗ 10 = 121.74 ∗ 10 �

�!"#$ !"#$! = 122��

Calculations of the mRNA(Sirt1) after the first purification:

To calculate the theoretical mole, the limiting factor was decided. CTPs was the limiting factor and for each 10 CTPs on mRNA(Sirt1) is created.

� �� % = !"#!$%&!'( ∗ 100 �!!!"#!$%&'(

!! �!"# = 100 ∗ 10 �

!! �!"# = 200 ∗ 10 �

!! !! !! �!"# = �!"# ∗ �!"# = 100 ∗ 10 ∗ 200 ∗ 10 = 2 ∗ 10 ��

!! �!!!"#!$%&'( = �!"# = 2 ∗ 10 ��

!! �!"#!$%&!'( = �!"#$(!"#$!) = 14.37 ∗ 10 ��

14.37 ∗ 10!! �� % = ∗ 100 = 0.72% 2 ∗ 10!!

The final yield after the first purification was 0.72%.

Calculations after the second purification:

�!"# = 0.086 � � = 263300 !"#$(!"#$!) (�� ∗ ��)

� = 1 ��

�!"# 0.086 !! �!"#$(!"#$!) = = = 0.33 ∗ 10 � �!"#$ !"#$! ∗ � 263300 ∗ 1

!! !! �!"!"(!"#$!) = �!"#$(!"#$!) ∗ �!"#$ !"#$! = 8471.5 ∗ 0.33 ∗ 10 = 2.77 ∗ 10 �

�!"#$ !"#$! = 2.77��

Calculations of the yield after the second purification:

� �� % = !"#!$%&!'( ∗ 100 �!!!"#!$%&'(

0.33 ∗ 10!! �� % = ∗ 100 = 0.02% 2 ∗ 10!!

The final yield after the second purification was 0.02%.

10. APPENDIX C - Educational material

Bilaga 1. Lärarhandledning

Framtidens genteknik

- Jakten på evigt liv

Utbildningsmaterial för gymnasieskolan

Materialet är skapat som en del av Mona Farshchians examensarbete

Lärarhandledning

Denna handledning är till för dig som ska använda dig av utbildningsmaterialet vid namn Framtidens genteknik-Jakten på evigt liv. Utbildningsmaterialet utgår från ett SNI-fall som innebär att samhällsfrågor behandlas i det naturvetenskapliga klassrummet. Detta material kommer ge dig som lärare instruktioner om hur uppgiften ska gå till då den ska genomföras av elever. SNI-fallet kan utnyttjas som komplement i undervisningen och kan hjälpa läraren att väcka elevernas intresse genom att få de att diskutera samhällsfrågor utifrån elevernas egna erfarenheter.

Utgångspunkt En stor fråga som diskuteras i dagens samhälle är genmanipulation, där forskare har kommit så långt i utvecklingen att de kan substituera arvsmassa i djur, grödor och växter för att kunna förbättra egenskaper. SNI-fallet som skapats bygger på ett filmklipp där människan och gentekniken diskuteras, där de visar hur världen kan se ut om 50 år då genmanipulation på människor kan komma att börja. Gentekniken har bidragit med olika möjligheter för människan, bland annat att skapa mat till den allt mer växande befolkningen på jorden och bota svåra sjukdomar. Filmklippet tar upp de etiska frågorna som kan förekomma kring genmanipulation. Ska vi skapa bättre mat och bota olika sjukdomar genom att göra olika förändringar i arvsmassan? Var går gränsen för vad som är rätt och fel?

Genmanipulation är ett hett ämne som debatteras och det filmklipp som rekommenderas i detta material ingår i en serie av avsnitt som alla är relaterade till genmanipulation, utifrån olika synvinklar. Det finns även en mängd olika artiklar som tar upp genmanipulation i form av debatter, men även organisationer såsom Greenpeace som är emot detta finns att hitta på internet.

Fallbeskrivning SNI-fallet behandlar som tidigare beskrivet genmanipulationens för och nackdelar. Du som lärare ska kort introducera ämnet och beskriva för eleverna att de kommer bli tilldelade olika roller som de ska argumentera för, där hälften av klassen ska vara för genmanipulation exempelvis forskare och hälften av klassen ska tillhöra organisationen Greenpeace som är emot genmanipulation. Eleverna ska ha klart för sig att de själva ska ta reda på information utöver filmklippet och skapa argument enligt den roll de blivit tilldelade. Detta beskrivs i en utförligare beskrivning längre ner.

Elevmaterialet delas ut då eleverna förstått instruktionerna till uppgiften, här beskrivs uppgiften utförligare och eleverna kan använda det som underlag för sin argumenterande uppgift. Hur uppgiften ska examineras hittas lite längre fram. När det är dags för argumentationen i klassrummet kan läraren dela in eleverna i grupper om fyra elever, där två elever är för genmanipulation och två elever är emot så en mer utförligare diskussion kan genomföras. Efter lektionen kan alla elever lämna in sina enskilda argument till läraren. Hela gruppen ska även skriva en kort sammanställning om vad de kommit fram till under sin argumentation. Vad är för-och nackdelarna med genmanipulation? Ska vi bota alla sjukdomar och leva ett evigt liv? Ska vi kunna designa våra barn? Är det rätt att genomföra tester av läkemedel på djur? Detta är frågor som kan diskuteras på många olika nivåer och är globala frågor som eleverna ska ta ställning till.

Naturvetenskapligt innehåll Den fråga som ska bearbetas i detta SNI-fall har klara kopplingar till naturvetenskapens utveckling, samt forskning inom kemiämnet samtidigt som det är en stor samhällsfråga som kräver diskussion. I ämnesplanen för ämnet kemi 2 på gymnasiet är några av kursmålen direkt relaterade till detta SNI fall:

• Det genetiska informationsflödet, inklusive huvuddragen i de biokemiska processerna replikation, transkription och translation. • Frågor om etik och hållbar utveckling kopplade till kemins olika arbetssätt och verksamhetsområden. • Kunskaper om kemins betydelse för individ och samhälle.

I fallet kommer eleverna få lära sig om hur genmanipulering sker, och genom den argumentationsuppgift de ska förbereda kommer de behöva diskutera etiska frågor som rör individen men även hur det påverkar hela samhället i stort. Detta står även beskrivet som ett av kemiämnets syften på skolverkets hemsida:

”Genom undervisningen ska eleverna ges möjlighet att utveckla ett naturvetenskapligt perspektiv på vår omvärld. I undervisningen ska aktuell forskning och elevernas upplevelser, nyfikenhet och kreativitet tas till vara. Undervisningen ska också bidra till att eleverna, från en naturvetenskaplig utgångspunkt, kan delta i samhällsdebatten och diskutera etiska frågor och ställningstaganden.”(Skolverket)

Samhällsvetenskapliga aspekter SNI-fallet kan diskuteras utifrån flera olika aspekter. Genmanipulation kan i många avseenden ses som en positiv utveckling, att maten vi äter kan bli bättre genom att vi förändrar i dess arvsmassa, att vi kan rädda världssvälten, att vi kan skapa nya läkemedel och bota sjukdomar eller förhindra att människan åldras. Men var går gränsen? Vems ansvar är det att avgöra om genmanipulation ska få genomföras eller inte? Vilka etiska frågor kan kopplas till detta på individnivå och samhällsnivå? I många länder genmodifieras redan mat för att det ska få förbättrade egenskaper samtidigt som det ska vara billigare att ta fram. Detta fall kan som nämt tidigare diskuteras utifrån olika synvinklar och genmanipulation är något som kan appliceras på allt levande och det mest drastiska är om det ska genomföras på människan, vilket kan leda till stora etiska diskussioner.

Intressekonflikt Detta fall kan skapa många intressekonflikter då man ser på det från olika synvinklar. I klippet som eleverna ska se visas en familj som genom genmanipulation lyckas rädda sitt sjuka barn och filmen visar att genteknik är en självklarhet för den familjen. Men vad händer då alla människor börjar använda genmanipulation för att skräddarsy sina barn till att få de ultimata egenskaperna eller det bästa utseendet? Hur kommer världen att se ut om ingen människa åldras och hur hanterar vi en överbefolkad värld?

Det diskuteras även mycket kring genmodifierad mat och om denna mat är farlig för människans hälsa. Utöver detta så utförs alla tester av läkemedel först på djur som har blivit genmanipulerade för att likna människan. Är det rätt att göra detta mot djuren för att människan ska klara sig bättre?

Det finns mycket att diskutera kring ämnet genmanipulation och på samma gång mycket att lära sig kring ämnet eftersom det berör hela samhället.

Vad eleverna ska använda sina kunskaper till De kunskaper som eleverna förväntas lära sig då de arbetar med detta SNI-fall är att själva tar fram information från olika källor och lära sig att vara källkritiska. Genom att eleverna blir tilldelade en roll, antingen som en person som är positiv till genmanipulation eller som en person som är negativ till genmanipulation, får eleverna en bredare bild av av ämnet och kan efter argumentationsuppgiften ta ställning i frågan.

Mål för fallet Det eleverna ska kunna efter detta arbete är • Söka information kring en specifik uppgift och vara källkritisk till det som hittas. • Biokemiska förlopp kring genmanipulation, arvsmassan ersätts med den förbättrade egenskapen. • Ta ställning i kritiska och svåra frågor såsom genmanipulation och dess för-och nackdelar.

Resurser Kring ämnet genmanipulation finns det både många filmklipp och tidningsartiklar, det klipp som rekommenderas kan hittas på youtube och heter Människan och Generna.1(b) det skräddarsydda barnet. Detta filmklipp ingår i en serie av filmklipp som behandlar olika delar inom genmanipulation, vilka också kan vara intressanta att titta på för att få en bredare uppfattning kring ämnet. Till de olika roller som eleverna blir tilldelade kan material hittas på Greenpeace hemsida, Forskningsartiklar och även debattartiklar ifrån tidningar. Två olika artiklar kan även delas ut som ger perspektiv över de olika argumenten kring genmanipulation http://www.svd.se/opinion/brannpunkt/genmanipulation-ger-inte-ett-uthalligt- jordbruk_6951821.svd http://www.svt.se/nyheter/vetenskap/forskarattack-mot-eu-landernas-gmo-regler

Länken nedan kan även användas för att ge en fördjupad inblick i de olika frågor som tas upp kring genmanipulation. http://henrikbranden.se/svara-fragor/

Rapportering och bedömning Rapporteringen kan ske genom att eleverna förbereder sina argumentationer och lämnar in dessa till läraren. Eftersom eleverna ska genomföra argumentationen i grupper om fyra elever, så kan en kort sammanställning av det de kommit fram till skrivas ner och lämnas in till läraren. Detta kan vara bra eftersom det visar att eleverna tagit del av vad hela gruppen argumenterat om och kan ta ställning till frågan tillsammans, genom att skriva ner några punkter.

Framtidens genteknik- Jakten på evigt liv

I och med att forskningen utvecklas så hittar forskare hela tiden nya möjligheter till att förbättra mat, djur och även människor. Genmanipulation innebär att man ersätter arvsmassan med förbättrade egenskaperna, ett exempel är genmanipulerad mat. Men det tar inte slut där utan alla tester av läkemedel genomförs på djur som har fått människoliknande egenskaper för att se om det fungerar eller inte. Genmanipulation blir allt vanligare och är en stor fråga som diskuteras i hela världen.

Uppgift När du har sett filmklippet om hur genmanipulation kan komma att användas till att förbättra egenskaper hos människor kommer du att bli tilldelad en roll. Du ska förbereda dig på att argumentera kring genmanipulation utifrån den rollen du blivit tilldelad. Antingen är du en person som är för genmanipulation(Forskare) eller en person som är emot genmanipulation(Medlem i Greenpeace). Ta reda på information kring ämnet och skriv ner egna argument som den roll som du blivit tilldelad skulle stå för. Du kan fundera över fördelar/nackdelar som finns med genmanipulation genom att ge olika exempel på då det förekommit och varför det är bra/dåligt.

Förbered dig på att kunna argumentera för nedanstående frågor utifrån den roll du har blivit tilldelad. • Vem ska bestämma om genmanipulation ska få genomföras? • Varför genomförs genmanipulation? • Kan världssvälten försvinna med genmanipulation och hur kan det påverka på individ/samhälls/globalnivå? • Ska vi människor få designa våra barn för att minska risken för svåra sjukdomar, bli starkare, snyggare och få bättre personliga egenskaper? • Tester av nya läkemedel sker på djur som har blivit genmanipulerade är det rätt eller fel? Finns det något annat sätt att genomföra detta?

Bilaga 2 Enkätundersökning

Enkätundersökning - Elever

Denna enkätundersökning riktar sig till dig som har arbetat med uppgiften Framtidens genteknik- jakten på evigt liv. Studien genomförs av Mona Farshchian masterstudent från Kungliga Tekniska Högskolan.

Vänligen kryssa i vilken/vilka av dessa förmågor du anser att du haft möjlighet att utveckla under ditt arbete med uppgiften Framtidens genteknik-jakten på evigt liv.

Kunskaper om kemins begrepp, modeller, teorier och arbetsmetoder samt förståelse av hur dessa utvecklas.

Förmåga att analysera och söka svar på ämnesrelaterade frågor samt att identifiera, formulera och lösa problem. Förmåga att reflektera över och värdera valda strategier, metoder och resultat.

Förmåga att planera, genomföra, tolka och redovisa experiment och observationer samt förmåga att hantera kemikalier och utrustning.

Kunskaper om kemins betydelse för individ och samhälle.

Förmåga att använda kunskaper i kemi för att kommunicera samt för att granska och använda information.

Tack för din medverkan!

Bilaga 3 Kvalitativ intervju

Intervju med lärare efter att materialet testats

• Hur tyckte du om uppgiftens upplägg? • Varför?

• Brukar ni göra denna typ av uppgifter? • Hur brukar det gå?

• Vilka förmågor tycker du att uppgiften speglar? • Varför?

• Vilka förmågor tycker du inte att uppgiften speglar? • Varför?

• Hur ser du på att ta in samhällsvetenskaplig frågor i naturvetenskapen?

• Hur tror du att eleverna reagerade på uppgiften? • Varför?

• Finns det något du skulle göra annorlunda? • Varför?