Why adapter ligation? Ligases. Oligonucleotide ligases. Definition of ligase

Transcription

1 Why adapter ligation? Ligases Introduction to s in general, and RA 1 / RA 2, truncated in particular mira bacterial mra -P unknown sequence 3 -H -PPP unknown sequence 3 -H 3 adapter LIGASE catalyzed known sequence 3 -P unknown sequence known sequence 3 -PPP unknown sequence known sequence 3 Definition of Wikipedia: ( )a ( ) is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group pendant to one of the larger molecules. ligonucleotide s acceptor 3 -H -P donor 3 ATP AMP PP i 3

2 1st step: ADEYLATI 2nd step: BD FRMATI ATP AMP PP i -P 3 3 BD FRMATI - chemistry ligonucleotide s adenylated donor (DA) ligated oligo Ligase T4 RA 1 (primary) Substrate ss RA C H 2 H H H H - P P - P - 3 terminus of acceptor (RA) G P - G P - Rnl2, truncated 0 C, overnight, -AMP P - P - C H G P - G P - T4 RA 2 T4 RA 2, truncated (mutated) T4 DA dsra ssra, preadenylated donor dsda

3 T4 RA 1 substrates acceptorrequirements: 3 H last fewnucleotides(3 ) HAVE T bera DA or RA RA3 -H in principle, the nucleotides before can be DA pra(in vitro transcribed): RA, 3 H donor: canbeda morestable -P DA or RA 3 phosphate(can be introduced enzymatically prior to ligation) adapteroligonucleotide: chemicallysynthesizedda, phosphate, 3 HEX-dye(purchased from IBA) RA 2, truncated substrates acceptor requirements: same asra 1 in vitro transcribed pra donor: canbeda DA or RA RA3 -H AMP -P DA or RA 3 adenylated(1st stepofligationcannotbe performed by this mutated enzyme!) adapteroligonucleotide, same asfort4 RA 1, pre-adenylated[by us] Preadenylation Preadenylation Chemical reaction: ribose H H H H phosphate - P - P - P G - P G H H 2 imidazole P - adenine G 50 C, 1.5 h P - ImpA G P - P - phosphorylated DA oligo preadenylated DA oligo Tasks 1. Tuesday, ctober 20: 1. Preadenylation of the adapter oligonucleotide 2. store the mixture at-20 C overnight 2. Wednesday, ctober 21: 1. prepare 12% denaturing PAGE 2. loadsamples(adenylationmixture) loading buffer with dye 3. run PAGE 4. scan PAGE if time, quantify bands 5. excise bands 6. start elution, which will continue overnight 3. Thursday, ctober 22: 1. Isopropanol precipitation Preparation read instruction for preadenylation in script calculate the missing values ERRR in script: after 1.5 h, add5 µl T 50 µl of ImpA solution CHAGE: incubation in PCR-tubes instead of Eppendorf tubes read instruction for PAGE in script (don t worry, we will show/help you) calculate missing values read instruction for Isopropanol precipitation in script

4 Samples for adapter ligation adapter buffer final [ATP] groups Rnl2, tr adenylated Rnl2, tr 0 mm 1-6 Rnl2, tr non-adenylated Rnl2, tr 1 mm (add) 1 & 2 Rnl1 non-adenylated Rnl1 1 mm (in buffer) 1-6 Rnl1 non-adenylated Rnl2, tr 1 mm (add) 3 & 4 Rnl1 adenylated Rnl2, tr 0 mm 5 & 6 control experiments buffer Rnl2, truncated 1XT4 Rnl2 truncated Reaction Buffer: 50 mm Tris-HCl 2mMMgCl 2 1mMDTT ph 7.5@ 25 C buffer Rnl1 1XT4 RA Ligase 1 Reaction Buffer: 50 mm Tris-HCl 10mMMgCl 2 1mMATP 10 mm Dithiothreitol ph 7.8@ 25 C Adapter Ligation Adapter ligation scheme (real life) Task 1. Thursday, ctober 29: 1. adapter ligation with preadenylated adapter oligo(ra 2, tr) 2. adapter ligation with non-adenylated adapter oligo(ra 1) 3. let reactions run overnight 2. Friday, ctober 30: 1. store ligation mixtures at -20 C 3. Monday, ovember 2: 1. 12% PAGE of ligation mixtures 2. load 3. run 4. scan(hex & SybrGold) 5. stain with SybrGold 6. scan(sybrgold) 7. quantify Preparation read instruction for adapter ligation (RA 2, tr and RA 1) in script CHAGE: no ATP needs to be added to buffer for Rnl1 check which control experiment(s) your group is performing mira bacterial mra -P unknown sequence 3 -H -PPP unknown sequence 3 -H -P unknown sequence known sequence 3 AMP 3 adapter -P known sequence 3 RA 2, truncated -PPP unknown sequence known sequence 3

5 Continued ther possibilities -P unknown sequence known sequence 3 DA or RA known sequence RA PCR adapter DA or RA known sequence RA3 -H RA 1 ATP unknown sequence known sequence 3 sequen cing reverse transcription -P unknown sequence known sequence 3 RT -P unknown sequence known sequence 3 3 cda digest RA 3 cda RA 1 with changed conditions: DA-DA ligation ligation 1. ribotailing (add several (ribo)gs) with terminal-deoxynucleotidyl transferase (TdT), 2.ligate a doublestranded anchor with poly-c overhang using DA- Concept In vitro transcription Introduction purified polymerase DA template TPs (ATP, CTP, GTP, UTP) standardized conditions salt other molecules

6 Polymerases Templates phage polymerases: T7 RA polymerase SP6 RA polymerase T3 RA polymerase Expressed in E.coliand purified Promotor sequences: modifiedfrom 1: G; 2: G/A otherwise poor yield Plasmids(linearized by restriction digestion) PCR products(can be used directly) ss/dsda hybrid (annealed before transcription) 3 A T T A T G C T G A G T G A T A T C T A A T A C G A C T C A C T A T A G G c o d i n g s e q u e n c e 1 Where to put the T7-promotor? sense or antisense transcripts 3 Transcribed sequences Template: T7-promotor pra only Hammerhead construct: internal promoter 9 ntcomplementary to pra hammerhead ribozyme pra transcription Transcription conditions 4-5 mm TP concentration (each) MgCl 2 Spermidine (alternatively BSA) stimulates polymerase DTT (reduce disulfide bonds) 18/Spermidine-2D-skeletal.png 61/Disulfide_reduction_by_DTT-2.png

7 Use transcribe sequences into RA (for studies, etc.) introduce labelled nucleotides (e.g., radioactive labels α 32 P-TP introduce functional groups selection of catalytic RA (e.g., Diels-Alderase) initiator nucleotide: GMP functional group Protein production/purification Coding sequence on a plasmid, behind a Lacpromotor Selection by Ampicilline resistance Initiation by IPTG Cells pelleted Lysis by sonication Centrifuge down cell debris, DA, etc. Purification Purification Protein containsa His 6 -tag Tag complexes ickel ickel-column-chromatography Protein containsa His 6 -tag Tag complexes ickel ickel-column-chromatography 1. load isolated proteins

8 Purification Purification Protein containsa His 6 -tag Tag complexes ickel ickel-column-chromatography 1. load isolated proteins 2. wash away unbound protein Protein contains a His 6 -tag Tag complexes ickel ickel-column-chromatography 1. load isolated proteins 2. wash away unbound protein 3. elute bound protein with imidazole Purification Protein containsa His 6 -tag Tag complexes ickel ickel-column-chromatography 1. load isolated proteins 2. wash away unbound protein 3. elute bound protein with imidazole automated Concentration of fractions Using a membrane with small pores molecular weight cutoff Upon centrifugation, salt, water and other small molecules will pass through pores bigger molecules are retained big molecule pore membrane small molecule modified from: how-pore-sees_ gif

9 Desalting by size exclusion chromatography T7-RA-Polymerase Task 1. Tuesday, ctober 20: 1. Inoculate preculture with glycerol stock of E.coli 2. Wednesday, ctober 21: 1. Induction of T7-RA- Polymerase-production 2. Miniprep of T7-RA- Polymerase Plasmid from preculture 3. Analysis of plasmid on agarose gel 4. Pelletting induction culture 3. Monday, ctober Sonicate pellet 2. protein purification by column chromatography Preparation Safety lecture Working with GeneticallyModifiedrganisms Read instructions from script to get an overview of working steps T7-RA-Polymerase... continued Task 3. Monday, ctober Sonicate pellet 2. protein purification by column chromatography 3. SDS-PAGE of fractions 4. concentrate fractions 4. Tuesday, ctober Desalt by PD-10 column 2. Add glycerol and measure concentration 3. Use isolated T7-Polymerase for in vitro transcription 5. Wednesday, ctober % PAGE of transcription products Preparation Read instructions from script to get an overview of working steps Remark: Desalting and concentration might be interchanged depending on size of protein-containing fractions Possibly concentrate after desalting, depending on protein-content Read instructions for in vitro transcription