Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Nucleic Acids – Basic Concepts David Murray PhD UCD|Mater Clinical Research Centre UCD School of Medicine and Medical Sciences Mater Misericordiae University Hospital Dublin DNA and RNA are Nucleic Acids Outline • What are DNA and RNA ? • The Structure and Function of DNA and RNA • What is a Gene ? • What is a Genome ? DNA and RNA What's the Big Deal ? • • • • • Hereditary Genetics Importance of Genetics in Disease Predisposition Mutations Loss of Genetic Control What is DNA ? • Contained in the nucleus • Arranged in 22 chromosomes, plus two sex chromosomes • Two copies of each (46) • 99.9% identical to other humans, 98% to chimp! • Each cell; 6 feet of DNA – >billion miles of DNA in the body! • Therefore, very tightly packed The Structure of DNA and RNA • DNA (deoxyribonucleic acid) • RNA (ribonucleic acid) – What’s the Difference ? • Both composed of two different classes of nitrogen containing bases: – the purines and pyrimidines. The Purines • The most commonly occurring purines in DNA are adenine (A) and guanine (G) A G The Pyrimidines • The most commonly occurring pyrimidines in DNA are cytosine (C) and thymine (T) C T RNA • Contains the same bases as DNA with the exception of thymine. • Instead, RNA contains the pyrimidine uracil (U) • DNA : AGCT RNA : AGCU T U The building blocks… • Purines and pyrimidines form chemical linkages with pentose (5-carbon) sugars. • The carbon atoms on these sugars are designated 1', 2', 3', 4' and 5'. T A • It is the 1' carbon of the sugar that becomes bonded to the nitrogen atom at position N1 of a pyrimidine or N9 of a purine. • DNA precursors contain the pentose deoxyribose. • RNA precursors contain the pentose ribose (which contains an additional OH group at the 2' position) T A Base (Purine/Pyrimidine) + Pentose (Deoxyribose/Ribose) = Nucleoside Nucleosides The resulting molecules are called nucleosides and can serve as elementary precursors for DNA and RNA synthesis, in vivo. Acid Nucleotides • Before a nucleoside can become part of a DNA or RNA molecule it must become complexed with a phosphate group to form a nucleotide (then termed either a deoxyribonucleotide or ribonucleotide). • Nucleotides can posess 1, 2 or 3 phosphate groups, e.g. the nucleotides adenosine monophosphate (AMP), adenoside diphosphate (ADP) and adenosine triphosphate (ATP). • The phosphate groups are attached to the 5' carbon of the ribose sugar. Beginning with the phosphate group attached to the 5' ribose carbon, they are labeled α, β and γ phosphate. • It is the tri-phosphate nucleotide which is incorporated into DNA or RNA Nucleotide (dNTP) T dTTP Where N = A / G /C / T / U Polynucleotides • DNA and RNA are simply long polymers of nucleotides called polynucleotides. • Only the α phosphate is included in the polymer. It becomes chemically bonded to the 3' carbon of the sugar moiety of another nucleotide. • Phosphate ‘backbone’ is negatively charged. Where ‘Base’ = A,C,G or T Polynucleotides • The polynucleotide is connected by a series of 5' to 3' phosphate linkages. • Polynucleotide sequences are referenced in the 5' to 3' direction. • Typically, polynucleotides will contain a 5' phosphate and 3' hydroxyl terminal groups. Summary of Terms • DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) composed of nitrogen containing bases – Purines: adenine (A) and guanine (G) – Pyrimidines: cytosine (C) and thymine (T) (uracil (U) in RNA) • Link with pentose sugars (DNA: deoxyribose, RNA: ribose) to form nucleosides – Nucleosides complex with three phosphate groups (Nucleotides) – Polymers are incorporated into DNA/RNA (polynucleotides) Summary of Terms Base Nucleoside RNA/DNA (Triphosphate) Code Adenine Adenosine dATP A Guanine Guanosine dGTP G Cytosine Cytidine dCTP C Thymine Thymidine dTTP T Uracil Uridine dUTP U What is the structure of DNA? How is the structure related to function? History • 1950 – Primary chemical structure of polynucleotides was known (i.e. the 5'-3' phosphate linkage). • 1951 – Erwin Chargaff: • Experiment: To analyse DNA from a variety of species and determine the relative concentrations of individual pyrimidines and purines (A, T, C and G bases). • Result: Although different species had uniquely different ratios of pyrimidines or purines, the relative concentrations of adenine always equaled that of thymine, and guanine equaled cytosine. • Chargaff's Law: A=T, G=C History • 1953 – J.D. Watson and F.H.C. Crick: • Identified a hydrogen bonding arrangement between models of thymine and adenine bases, and between cytosine and guanine bases which fulfilled Chargaff's rule. • “Double Helix” _ G=C A=T Consequences • If G always paired with C, and T always paired with A, then either strand could be regenerated from the complementary information in the other strand. • The basis of the complementarity was hydrogen bonding, i.e. non-covalent interactions which could be easily broken and re-formed. • The information which DNA carried was within the unique base sequence of the DNA. • From the general interior location of the bases, it would appear that the double helix would have to dissociate in order to access the information. DNA Structure • Thousands of nucleotides are strung together by a phosphate-sugar backbone DNA Structure • Two strands of DNA twist around one another to form a double helix • ‘Twisted Ladder’ • Complementary base pairs form the rungs DNA Structure • Two nucleotide sequences running in opposite directions pair with one another. • Each adenine (A) pairing with a thymine (T) • And each guanine (G) pairing with a cytosine (C) Base Pairs (BP) 5' C-G-A-T-T-G-C-A-A-C-G-A-T-G-C 3' | | | | | | | | | | | | | | | 3' G-C-T-A-A-C-G-T-T-G-C-T-A-C-G 5' A Word on DNA function • Carries the blueprint for life • Duplication for new cells • Make proteins for biological functions: DNA • Prior to cell division, DNA is replicated before being it is passed on to daughter cells • The DNA within our cells contains the information for everything which occurs within each cell, – every action – every substance made – every event – every response – everything! The Genome The Genome • The complete set of information in an organism's DNA is called its genome • Carries the information for all the proteins the organism will ever synthesize. • Typical human cell – 6 feet of DNA – Written in the four-letter nucleotide alphabet that spells out the linear sequence of amino acids in a protein. – Carries instructions for ~ 30,000 different proteins Gene Expression What are Genes ? The Cell Library Chromosomes Books Genes Sentences • Approx 26,000 human genes • Made up of DNA • Coding regions of DNA Gene Structure • Like a sentence – beginning (Start) and end (Stop) • Ordered structure – not random bunch of nucleotides linked in some random order Transcription • The first step in gene expression • DNA (gene) is used as a template to synthesise RNA copy • Transcriptional Profiling • A specific gene specifies a polypeptide (protein) – The DNA is transcribed into message RNA (mRNA), which is translated into the polypeptide DNA TRANSCRIPTION RNA TRANSLATION Protein Translation • • • mRNA used as template to make proteins Occurs in ribosomes One codon corresponds to one amino acid • A specific gene specifies a polypeptide Gene 1 Gene 3 DNA molecule Gene 2 DNA strand TRANSCRIPTION RNA Codon TRANSLATION Protein Amino acid The Genetic Code • Specific DNA sequences code for specific amino acids Transcribed strand Mutations ? DNA Transcription RNA Start codon Translation Stop codon Faulty Protein Polypeptide Summary An Example Nucleotide sequence for Human BetaGlobulin gene. • Haemoglobin subunit • Caries information for amino acid sequence of one globulin subunit molecule. • Alpha globulin – another gene • Only one of two complementary strands of DNA shown • Written and read from left to right (from 5’ to 3’) like text. • DNA highlighted in yellow: regions that specify amino sequence for protein. Review • DNA and RNA – What they are – Structure and Function • Genes and Genomes • Transcription and Translation • Any Questions ? Nucleic Acids – Analytical Techniques David Murray PhD UCD Clinical Research Centre UCD School of Medicine and Medical Sciences Overview… • Analytical Techniques • RNA Techniques – Extraction – Analysis • Reverse Transcription – RNA → DNA • RTPCR – Reverse Transcription Polymerase Chain Reaction • Quantitative Real Time PCR • RNA interference (siRNA) • Introduction to Microarrays RNA Analysis Extraction Quantitation Quality Assessment Why Analyse RNA ? • • • • • Transcriptional Profiling Levels of mRNA expression mRNA: early step in gene expression Controlled step Variation between different cases – Normal Vs Disease – Responders Vs Non-Responders ANALYSIS OF GENE EXPRESSION 5’ DNA (gene): 3’ PROMOTER exon 1 intron exon 2 transcription 3’ 5’ RNA (1o Transcript) RNA processing Polyadenylated RNA analysis 5’ mRNA 3’ AAAAAA m7GpppN Note: Non coding Introns are not included in mRNA molecule translation protein RNA Analysis RNA RT-PCR Quantitative PCR Microarray Analysis •Comparison of mRNA expression profiles between two states; •Disease Vs Normal •Treated Vs Untreated •Primary Vs Mets Working with RNA RNA is more susceptible to degradation than DNA The 2´ hydroxyl groups adjacent to the phosphodiester linkages in RNA are able to act as intramolecular nucleophiles in both base- and enzyme-catalysed hydrolysis. DNases require metal ions for activity and so can be inactivated with chelating agents e.g. EDTA RNases bypass the need for metal ions by taking advantage of the 2´ hydroxyl group as a reactive species. Problems with RNases • RNases – single-strand specific endoribonucleases – resistant to metal chelating agents – can survive prolonged boiling or autoclaving • But… – relies on active site histidine residues for activity – Therefore, it can be inactivated by the histidinespecific alkylating agent diethyl pyrocarbonate (DEPC). Avoiding ribonucleases Exogenous Introduced during working procedures Eliminate through good working practices Endogenous Released by cells or tissue during extraction Eliminate through use of inhibitors of RNase activity Working with RNA – Dos and Don’ts Always wear gloves - Skin is an abundant source of ribonucleases. Prepare solutions for RNA work using autoclaved glassware, then autoclave the solutions after they are prepared. Better still use disposable plastic ware if possible. If possible use pre-sterilized water. Use separate solutions for RNA work and only use them for RNA. DEPC treatment of water isn’t always necessary. Autoclaving water and solutions can sometimes be more effective in removing RNases than chemical treatment. Working with RNA – Dos and Don’ts If you do need to treat your solutions with DEPC: 1.make your solution 0.1% DEPC (500 µl in 500 ml H2O) 2.shake it well 3.keep it overnight at RT 4.autoclave Take care! DEPC is highly carcinogenic. Use a fumehood! Working with RNA – Dos and Don’ts Maintain a separate area for RNA work that has its own set of pipettes. This is especially important if your work requires the use of RNase A (e.g. plasmid preps). Sterile, disposable plasticware can safely be considered RNase-free and should be used when possible Use RNase away or RNase zap!! RNA Extraction …an example TRI ReagentTM • • • • • Sigma (Cat# T9424) RNA, DNA and Protein extraction Cell/Tissue lysis Liquid separation Quick and Effective Extraction from Cells in Culture • 80 – 100% Confluent T75 (~1 x 107 cells) • Remove all media, wash twice with PBS (saline) • Add 1ml TRI REAGENT (cover all cells) – Scale Down/Up for other culture vessels • 10 min at Room Temp (RT) • Remove to sterile microfuge tube Extraction from Tissue • Remove tissue from RNAlater into sterile microfuge tube. • Add 1 mL TRI REAGENT • Homogenise at 15,000 rpm for 1-2 min. • Wash Tip between samples in 100% Ethanol, then 0.1 % DEPC. Chloroform Separation • Add 200 µL (0.2 ml) Chloroform (fumehood!) • Mix well (vortex), and stand at RT for 15 min • Centrifuge at 12,000 x g (MAX!) for 15 min at 4oC • 3 layers: – Upper (aqueous): RNA – Middle (interphase): Protein – Lower (organic): DNA • Remove upper phase to fresh microfuge tube. Propanol Precipitation • Add 500 µL Ice-Cold Isopropanol and mix • Stand on Ice for 10 min • Centrifuge at 12,000 x g for 10 min at 4oC • Pellet ? • Remove Isopropanol • Add 1 ml 75% Ethanol and vortex • Centrifuge at 7,500 x g for 5 min • Remove Ethanol and allow to air dry (10 min) • Resuspend in 10 – 50 µL 0.1% DEPC (60oC 10 min) RNA Quantitation RNA Quantitation • UV Spectroscopy • 1/100 dilution of RNA – 5 µL RNA in 495 µL 0.1 % DEPC • Absorbance at 260nm and 280nm – Quartz cuvette – Blank with 0.1% DEPC RNA Quantitation Calculation • A260 = 1.0 (40 µg/mL) • Concentration (µg/µL) = A260 x 40 x 100 (diln. factor) 1000 (mL → µL) • Or Simply: A260 x 4 = Concentration (µg/µL) • A260/A280 Ratio: RNA Quality/Purity (≈ 1.8) – Higher: Organic Contaminants – Lower: Protein Contaminants RNA Quantitation Calculation • Eg – 1/100 dilution of RNA – Absorbance Values: • 260nm 0.456 • 280nm 0.250 – Concentration: • (0.456 x 40 x 100)/1000 • 0.456 x 4 = 1.824 µg/µl – Purity • 0.456/0.250 = 1.8 (perfect!) Newer technologies : BioAnalyzer NanoDrop Next? Assessment of RNA Quality Agarose Gel Electrophoresis • To assess Quality of RNA – Extent of degradation • Also used to as standard method for analysing, identifying and purifying fragments of DNA (later). “Electrophoresis” • A technique used to separate and sometimes purify macromolecules especially proteins and nucleic acids – based on their difference in size, charge or conformation. • When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. • In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. V I “Electrophoresis” • Nucleic acids are electrophoresed within a matrix or "gel". • The gel is cast in the shape of a thin slab, with wells for loading the sample. • Agarose is typically used at concentrations of 0.5 to 2%. • The higher the agarose concentration the "stiffer" the gel. • Agarose gels are extremely easy to prepare: simply mix agarose powder with buffer solution (TAE/TBE), melt it by heating, and pour the gel. It is also non-toxic. • The gel is immersed within an electrophoresis buffer (same as above) that provides ions to carry a current and it also maintains the pH at a relatively constant value. RNA Electrophoresis 1. 2. 3. 4. The agarose gel with three slots (S). Injection of RNA sample into the first slot. Injection of samples into the second and third slot. A current is applied. The RNA moves toward the positive anode due to the negative charges on its phosphate backbone. - + Gel System Procedure • Clean Gel System with RNase Inhibitor Spray • Mix 50ml 10X TAE (Tris Acetate EDTA) buffer with 450ml DIW (De-ionised Water) = 1X TAE • 0.5g Agarose in 50ml 1X TAE Buffer – 1% (w/v) solution/gel – Microwave until dissolved (1-2min @ 650W) • Pour into casting tray (with combs) and allow to cool/solidify Procedure • Analyse 2µg RNA by electrophoresis – 2/concentration (µg/µl) – Eg: • 1.824 µg/µl • 2 µg in ~ 1 µl • Mix with 1 µl DEPC and 0.5 µl RNA loading buffer • Heat 65oC 10 min then chill on ice • Submerge gel in 1X TAE (running buffer) • Load RNA (2.5 µl) on gel and run at 100V Procedure • Remove gel after ~ 40min (blue of buffer almost at end of gel) • Visualise under UV light • Visible ribosomal subunits indicate intact RNA – 1 Degraded – 2,3 Good Quality RT-PCR Reverse Transcriptase Polymerase Chain Reaction The basics… • Interested in gene expression (mRNA) • Levels of mRNA (transcripts) • Comparison between 2 states (normal and disease) • mRNA (1-5% of total RNA) • We use PCR (DNA Technique) – More on that later • Must Convert RNA to DNA – How ? Reverse Transcription • mRNA molecule is copied into a double stranded DNA compliment (cDNA) • Reverse transcriptase – enzyme that performs this. • Used naturally by retroviruses to insert themselves into an infected organism's DNA genome • cDNA contain coding regions only (exons) Reverse Transcription (RT) • mRNA Template • ‘Priming’ – polyA mRNA isolated from total RNA using oligo dT primer • Polynucleotide of T’s – Initiates synthesis • First strand of cDNA synthesised using Reverse Transcriptase (RT) enzyme – Adds complimentary nucleotide bases to mRNA to make cDNA cDNA is then used as a template in PCR PCR • Polymerase Chain Reaction – Technique for Targeted DNA Amplification – Starting material ('target sequence’); • A gene or segment of DNA (cDNA in our case) – Target sequence can be amplified a billion fold in a matter of hours • PCR allows one to take a specimen of genetic material, even from just one cell, copy its genetic sequence over and over, and generate a test sample sufficient to detect the presence or absence of a specific virus, bacterium or any particular sequence of genetic material PCR Applications • Widely used in molecular biology • Specific Amplification • Assuming sequence of target is known; – Viral Detection • HIV can be quantitated – Screening genes for mutations – Detecting gene expression – Detection of food pathogens – Forensic identification The PCR Reaction • Template – Target DNA that Primers will bind • Primers – Bind target sequence, making the reaction specific • Taq – enzyme which carries out the amplification reaction – extends the primers from their binding-sites on the target along the template • Buffer – Contains a salt (KCl) and MgCl (cofactor for Taq) • Nucleotides – A,T,G and C – Deoxyribonucleotide triphosphates (dNTPs) – DNA building blocks • Water – High ‘PCR’ grade PCR: Practicalities • Always wear gloves • All reagents must be thawed and mixed completely before use • Typical Reaction Mix (50µl); – 37.5µl sterile water – 5µl 10X Buffer – 1µl 10mM dNTP mix – 0.5µl Taq (5U/µl stock) – 1µl Primer 1 & 1µl Primer 2 (10 pmol/ul) – 5µl Template (cDNA) The PCR Procedure • Entire genomic double stranded DNA is heated (denatured) • Primers (DNA oligonucleotides) – flank the nucleotide sequence of the gene – synthesised chemically – Prime DNA synthesis on single stranded DNA • In vitro DNA Synthesis catalysed by DNA polymerase • Primers remain at 5’ end of new DNA fragments The PCR Cycle • Initial Cycle: 1min @ 95oC • Followed by 40 cycles of following; – Denature: 1min @ 95oC – Anneal: 1min @ 50-60oC (depends on primer) – Elongate: 1min @ 72oC • Final extension: 10min @ 72oC PCR Links • Calculate Annealing Temperature – http://www.bioinformatics.vg/bioinformatics_to ols/oligo2002.shtml • Primer Design – http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi – http://www.basic.nwu.edu/biotools/oligocalc.ht ml The Thermocycle The PCR Procedure PCR: DNA Amplification Agarose Gel Electrophoresis • Analysis of PCR product • Mix 50ml 10X TAE (Tris Acetate EDTA) buffer with 450ml DIW (De-ionised Water) = 1X TAE • 0.5g Agarose in 50ml 1X TAE Buffer – 1% (w/v) solution/gel – Microwave until dissolved (1-2min @ 650W) • Add 1µl 10mg/ml Ethidium Bromide and mix – Interacts with Nucleic Acids – Fluorescent Complex – Visible under UV • Potent Mutagen – Fumehood, Lab Coat, Safety Glasses, Gloves – Spills: Absorbed and Decontaminated with soap and water • Pour into casting tray (with combs) and allow to cool/solidify Running the Gel • Submerge gel in 1X TAE (running buffer) • Mix 3µl PCR reaction with 3µl loading buffer and load onto gel • Mix 3µl 100bp DNA ladder with 3µl loading buffer and load onto gel • Run at 100V • Remove gel after ~ 40min (blue of buffer almost at end of gel) • Visualise under UV light • Dispose Gel in Yellow Biohazard Bin DNA Electrophoresis 1. The agarose gel with three slots (S). 2. Pipette DNA ladder into the first slot. 3. DNA ladder loaded. loading of samples into the second and third slot. 4. A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone. 5. Small DNA strands move fast, large DNA strands move slowly through the gel. DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel. 6. The DNA is spread over the whole gel. The electrophoresis process is finished. Real Time PCR Traditional PCR – Limitations • • • • • • Qualitative not Quantitative Gel Required End point detection 4/5 hrs until result Non numerical Non Automated Real Time PCR • Monitor Amplification in Real Time • Measure the kinetics of the reaction in the early phases of PCR • Quantitative and Qualitative • 30/40 min until result • Numerical output • Automated PCR Phases Reaction components being consumed. Reaction is slowing. Doubling of product at every cycle. Area for Real Time Detection Reaction has stopped. No more products are being made. Real Time PCR Instruments SYBR Green Chemistry • A dye that binds the Minor Groove of double stranded DNA. • Increasing the intensity of the fluorescent emissions. • As more double stranded amplicons are produced, SYBR Green dye signal will increase. • Increase in fluorescence directly proportional to increase in amplicons (amplified product) produced, which is proportional to the amount of target template present initially. SYBR Green Chemistry Amplification Curve Real Time PCR • Pro – Sensitive – No gel • Con – Expense – Hardware Real Time PCR Links • Good Article – http://dorakmt.tripod.com/genetics/realtime.html • Troubleshooting – http://www.eurogentec.com/module/FileLib/GRTTSGCUST-0304-V4.pdf Microarrays Microarray Analysis Monitor the activity of thousands of genes simultaneously Compare activity of one gene in many samples. Compare activity of many genes in one sample. Take a photo of genes in action! • Thousands of genes and their products, in a living organism function in a complicated and orchestrated way. • Traditional methods in molecular biology generally work on a "one gene in one experiment“. – Limited throughput. – “Whole picture" of gene function is hard to obtain. • Microarrays – Monitor the whole genome on a single chip – Better picture of the interactions among thousands of genes Base-pairing or hybridization (A-T and G-C for DNA) (A-U and G-C for RNA) Underlining principle of DNA microarrays What is an Array ? aka Genechips aka BioArrays aka Biochips aka Genomechips • • • Microarrays involve the immobilization of defined nucleic acids sequences (probes) on a solid support… Subsequent binding of target sequences complementary to these nucleic acids to measure gene expression levels. The DNA sequences at each probe represent important genes (or parts of genes) • 1.28 x 1.28 cm glass/silicon wafer • • 24 x 24 µm probe site (≈ 500,000 probes) Lengths of DNA up to 25 nucleotides long GeneChip Applications of DNA Microarrays • DNA Microarrays are used to study gene activity (expression) • What proteins are being actively produced by a group of cells? • • • “Which genes are being expressed?” Compare expression levels How? • When a cell is making a protein, it translates the genes (made of DNA) which code for the protein into RNA used in its production • The RNA present in a cell can be extracted • If a gene has been expressed in a cell • • RNA will bind to “a copy of itself” on the array RNA with no complementary site will wash off the array • The RNA can be “tagged” with a fluorescent dye to determine its presence • DNA microarrays provide a high throughput technique for quantifying the presence of specific RNA sequences Control Disease Cells/ Tissues mRNA cRNA Hybridisation Expression Comparison The Process Poly-A RNA Cells AAAA 10% Biotin-labeled Uracil Antisense cRNA IVT L L L (In-vitro Transcription) cDNA Fragment (heat, Mg2+) Labeled fragments L L L Hybridize Wash/stain Scan Affymetrix GeneChip Techonology Hybridization and Staining Biotin Labeled cRNA GeneChip Hybridized Array L L + L L L L L + L L L L L SAPE Streptavidinphycoerythrin The Result •A light source scans the array, causing the dyes to fluoresce •The glow is picked up by a sensor and is used to determine the relative abundance of the RNA •This information must be processed to determine the level of activity for each expressed gene Data Clustering Array Applications • Basic Understanding • Arrays can take a snap shot of which subset of genes in a cell is actively making proteins • Medical diagnosis • Used to determine if a person’s genetic profile would make him or her more or less susceptible to drug side effects • Used to distinguish between similar diseases or to define previously unknown subsets within a disease. • Drug design • Translate the human genome results into new products • Must figure out what the genes do, how they interact, and how they relate to diseases. Microarray Links •Affymetrix –http://www.affymetrix.com RNA interference (siRNA) Outline 1. 2. 3. 4. 5. RNA interference (RNAi) – what is it? Mechanism of RNAi – an overview Meet the players Experimental Applications Therapeutic Applications what is RNA interference? •RNAi is a way to silence gene expression •to perform RNAi, dsRNA homologous to the targeted gene is made and then introduced into cells •mRNA with high sequence homology to the dsRNA may be silenced RNA Interference • Post-transcriptional gene silencing • First discovered in c. elegans and plants – Protective role: parasitic and viral resistance • Mammals – RNAi occurs – role??? How does RNAi work? RNAi works postranscriptionally…….. siRNAs have a defined structure 19 nt duplex 2 nt 3’ overhangs • siRNA binding • siRNA unwinding • RISC activation •(RNAi silencing complex) practical aspects of RNAi • biological research – defining gene function (gene knockout) – defining biochemical pathways • microarray screening of RNAi knockouts • therapeutic treatment – cancer – Infection Although silencing by siRNAs is transient, vectors can be made to express siRNAs in cells gene function analysis cell engineering in vitro drug target validation forward genetic screens future cell type of interest producer Short hairpin vector virus TISSUE CULTURE HIGH-THROUGHPUT tissue type of interest ES cell gene function analysis in vivo drug target validation gene interaction therapeutic testing tissue or time specific analysis of gene function future? INDUCIBLE KNOCK-OUT KNOCK-OUT/ -DOWN future? GENE THERAPY McManus and Conklin RNAi, 2003 siRNA Delivery • in vitro – Chemical transfection (Lipofectamine, Oligofectamine, TransIT-TKO, Siport Amine, Siport • in vivo – Intramuscular injection – Hydrodynamic transfection into mammals siRNA Therapeutics Therapeutic siRNAs siRNA target gene p53 mutant K-Ras BCR-ABL MDR1 C-RAF Bcl-2 VEGF PKC-α Disease Cancer Β-Catenin (Sioud, 2004) Therapeutic siRNAs siRNA target gene Disease HIV-Tat HIV-Rev HIV-Vif, -Hef HPV-E6 and –E7 HBV-S1, -S2, -S, -X CCR5, CXCR4 CD4 Viral Infection (Sioud, 2004) Therapeutic siRNAs siRNA target gene Fas receptor Caspase-8 TNF-α Disease Acute Liver Failure Sepsis (Sioud, 2004) References Hannon, G.J. (2002). RNA interference. Nature. 418; 244-251. (review) Agrawal, N. et al. (2003). RNA interference: biology, mechanism and applications. Microbiol. Mol. Biol. Rev. 67; 657-685. (review) Elbashir et al. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411; 494498. Fire, A. et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391; 806811. Agrawal, N. et al. (2003). RNA interference: biology, mechanism and applications. Microbiol. Mol. Biol. Rev. 67; 657-685. Tuschl, T. (2002). Expanding small RNA interference. Nature Biotech. 20; 446-448 Donze, O and Picard, D. (2002). RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Research. 30; e46 Hannon, G.J. (2002). RNA interference. Nature. 418; 244-251. McCaffrey, A.P. et al. (2002). RNA interference in adult mice. Nature. 418; 38-39. Shuey, D.J. et al. (2002). RNAi: gene-silencing in therapeutic intervention. DDT. 7; 1040-1046. Sioud, M. (2003). Therapeutic siRNAs. TIPS. 25; 22-28. Wall, N.R. and Shi, Y. (2003). Small RNA: can RNA interference be exploited for therapy? The Lancet. 362; 1401-1403 Extra Links • NCBI Human Genome: – http://www.ncbi.nlm.nih.gov/genome/guide/human/ release_notes.html • Human Genome Project: – http://www.genome.gov/ • General Protocols – http://micro.nwfsc.noaa.gov/protocols/protocols.ht ml