Základy molekulární biofyziky (in English) Part 6: Cellular Structural Biology - NA DNA as a drug target Single gene Multiple copies of mRNA Multiple x Multiple copies of protein RNA DNA 3 Environmentally Promoted Deformability: a fundamental difference between DNAand RNA RNA structure is insensitive to environmental conditions (A pH, A ion strengh, ion type, hydration, MC) DNA structure is sensitive to environmental conditions Examples of environmentally controlled polymorphism of NA anti-parallel basket 1 MC Kf crystal K" solution anti-parallel parallel basket hybrid-1 hybrid-2 anti-parallel basket (A pH, A ion strengh, ion type, MC, hydration) ... even helix geometry is controlled bv environment Vargason et al. PNAS 2001 Polymorphism as a source of "targets" Polymorphism as a source of "problems" in the process of drug development Architecture of telomeric in G-rich single stranded 3'-overhang - d(TTAGGG)n NMR Na+ antiparallel basket X-ray K+ parallel propeller NMR K+ 2-tetrad antiparallel basket Wang et al. Structure (I 993) Ambrus et al. Nucleic Acids Res. (2006) Parkinson et al. Nature (2002) Dai et al. Nucleic Acids Res. (2007) Lim et al. J Am Chem Soc. (2009) Structural Biology of NA - an issue How to recognize physiologically relevant structure Structural Biology of NA - methods X-ray diffraction Diffraction Process Diffraction Pattern from NSLS ... thus far, it is not possible to detect diffraction from single molecule© X-ray diffraction relies on monocrystal production X-ray diffraction & monocrystal production Crystal Screen™_HR2-110 Reagent Formulation Tube ■ Salt Tube u Buffer 0 Tube Precipitant ■ 1. 0.02 M Calcium chloride dihydrate n 1. 0.1 M Sodium acetate trihydrate pH 4.6 TT 1. 30% v/v (+/-)-2-Methyl-2,4-pentanediol 2. None 2. None 2. 0.4 M Potassium sodium tartrate tetrahydrate 3. None 3. None 3. 0.4 M Ammonium phosphate monobasic 4. None 4. 0.1 M TRIS hydrochloride pH 8.5 4. 2.0 M Ammonium sulfate 5. 0.2 M Sodium citrate tribasic dihydrate 5. 0.1 M HEPES sodium pH 7.5 5. 30% vA/ (+/-)-2-Methyl-2,4-pentanediol 6. 0.2 M Magnesium chloride hexahydrate 6. 0.1 M TRIS hydrochloride pH 8.5 6. 30% w/v Polyethylene glycol 4,000 7. None 7. 0.1 M Sodium cacodylate trihydrate pH 6.5 7. 1.4 M Sodium acetate trihydrate 8. 0.2 M Sodium citrate tribasic dihydrate 8. 0.1 M Sodium cacodylate trihydrate pH 6.5 8. 30% vN 2-Propanol 9. 0.2 M Ammonium acetate 9. 0.1 M Sodium citrate tribasic dihydrate pH 5.6 9. 30% w/v Polyethylene glycol 4,000 10. 0.2 M Ammonium acetate 10. 0.1 M Sodium acetate trihydrate pH 4.6 10. 30% w/v Polyethylene glycol 4,000 11. None 11. 0.1 M Sodium citrate tribasic dihydrate pH 5.6 11. 1.0 M Ammonium phosphate monobasic 12. 0.2 M Magnesium chloride hexahydrate 12. 0.1 M HEPES sodium pH 7.5 12. 30% vA/ 2-Propanol 13. 0.2 M Sodium citrate tribasic dihydrate 13. 0.1 M TRIS hydrochloride pH 8.5 13. 30% vN Polyethylene glycol 400 X-ray diffraction ... underlying assumptions Lowest energy structure is biologically active c Coordinates □ structure is independent of environmental conditions additives, hydration levels, temperature, MC, viscosity, concentration, ion type, ion strength,...... PDB statistics Exp. Method 1 Proteins Nucleic Acids 1 Protein- N A Other complexes X-ray 1 75 215 1 464 3 888 2 NMR | 8 737 1 030 | | 192 7_ El. Microsc. 42U 128 0 Hybrid 46 3 2 1 Other 148 ■ | 13 | Total 84 574 2 546 4216 23 Nuclear Magnetic Resonance Sample: water based solutions, [biomolecule] -50-3 mM, Te ~ 0 - 45 °C NMR spectroscopy ... underlying assumptions □ Lowest energy structure is biologically active (in principle NMR also allows determination of high energy states) O) c Coordinates NMR spectroscopy ... can be physiological, but... Ionic composition of: Intracellular space Extracellular space Ionic composition of buffers used for NMR studies of: potassium sodium magnesium I calcium I ion not specified DNA RNA Structural Biology - an issue Precision vs. accuracy Structural Biology - an issue conventional NMR as well as X-ray .. are only able to assess structure precision «• • • # • • • • X-ray ■ Resolution NMR spectroscopy ■ RMSD .. NOT its accuracy Dark secret of structural biology X-ray & NMR "shoot" without knowing where the target is ... assessment of structural accuracy presumes knowledge of reference structure Cellular Structural Biology ...on target shooting Cellular Structural Biology-a concept How to find a "target" In vitro structure & dynamics buffered solutions or crystalline state t in vivo structure & dynamics Complex environment of living cells Cellular Structural Biology - proteins - a history Proteins 2000 - in-cell NMR of proteins overexpressed in bacterial cells 2006 - In-cell NMR of proteins delivered into X. laevis oocytes 2009 - In-cell NMR of delivered proteins in mammalian cells; 1st high resolution structure of protein inside living cells 2011 ... - In-cell NMR of proteins overexpressed in yeast, insect cells, mammalian cells 2012 ... - In-cell EPR of proteins delivered in bacteria, X. oocytes Cellular Structural Biology - a history Nucleic Acids 2009 - in-cell NMR: DNA/RNA injected in X. laevis oocytes 2010 - In-cell EPR: DNA injected in X. laevis oocytes 2012 - In-cell spFRET: DNA in bacterial and mammalian cells all delivered n-cell NMR of nucleic acids NA delivery via mechanical injection Xenopus laevis Stage IV oocyte Hansel et al. J Am Chem Soc. 2009 Signals from NA vs. (friendly) cellular background Topology information from imino pattern To see the rest - isotopically labeled samples 13C ppm 80 88 b 1 ■ / 'I ■ k '"CV/C4' 15N ppm 145 8.5 8.0 7.5 7.0 1HPPm 150' -1- 5.0 4.0 -1-1- 6 5 -1-1-1 4 3 d e 90- 0 0 95- § fid * 0 10O imino amino 13.5 13.0 12.5 -1-1-1-r 8.0 7.5 7.0 6.5 ppm In-cell NMR: NA degradation (un)expected problem in cell in vitro ~ 5 hrs 8.5 8.0 7.5 7.0 8.5 8.0 7.5 7.0 8.5 8.0 7.5 7.0 In-cell NMR: NA degradation (un)expected problem 13.7 13.3 12.9 PPm - 30 min ---180 min ...... 360 min Chemical stabilization prevents NA degradation Phosphotioester moiety allows monitoring the NA backbone 62 58 54 50 PP™ In-cell ID 31P NMR spectra ' Ce||u|arbackground 62 58 54 50 PP™ Problem of intracellular localization Introduced DNA localizes in nucleus injected DNA nucleus cytosol Resolution limits the analysis of the polymorphs: Cellular lysates 12.0 11.0 ppm Resolution limits the analysis of the polymorphs: site-specific labeles Crude cellular GGGTTAGGGTTAGG TTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA I-1-1 I-I-I 13 12 11 10 ppm a good news: DNA/RNA (if there is any) can be recovered from cells 12.0 11.0 PPm 12.0 11.0 PPm cleared lysate (boiling) butanol washed 12.0 11.0 PPm 12.0 11.0 PPm Summary: in-cell NMR of NA ... NA can be studied inside eukaryotic cells at atomic resolution • <25 without isotopic labeling (imino H/secondary structure) • with isotopic labeling up to 70 nt • degradation can be diminished via chemical modification • experimental time-window < 3 (leakage, degradation) Application potential: • ale novo structure determination - limited (price-wise) • fold validation - YES • NA sensitivity to environmental factors - YES •DNA drug interactions - YES (Selgado & Mergny) Interpretation of in-cell NMR data: spectral fingerprinting a condition 1 condition 2 in vitro § § in vivo condition 3 1H[pp7i] 'H [ppm] YES Spectral fingerprints in vitro " in vivo YES Solve in vitro structure \ / N \ V NO Mimicking in vitro conditions? NO Solve in vivo structure Spectral fingerprinting: example Spectral fingerprinting: example Benchmarking of in-cell NMR spectra to NA motifs Base-pairing pattern (WC/Hoogsteen/i-motif) Imino - region 13 c H J d ISO Sugar pucker (C2-endo/C3-endo) ^> Glycosidic torsion angle (syn/anti) Stacking (A-like/B-like) Does "being in cell" means "being native"? Unnaturally high concentration of NA are introduced Injected cells propagate through meiosis injection of exogenous DNA progesteron * I G2 arrested oocyte meiosis I metophose meiotic interphase meiosis II metaphase Cells accommodate/tolerate introduced NA Towards structural biology under native conditions... X-ray NMR ln-cell NMR ln-cell spFRET In-cell single particle FRET DNA/RNA cell Fessl et al Nucl Acids Res. 2012 In-cell single particle FRET Interpretation based on rigid arrangement of tags might be biased Interpretation based on rigid arrangement of tags might be biased Interprobe distance In-cell A-DNA B-DNA Nucleic Acids Structural analysis under in vivo conditions DNA/RNA Ity i3C/i5N ^ty- fluorophorei *\ paramag. label %■ 2H cell 4*1 ln-cell NMR ln-cell spFRET ln-cell EPR n-cell Raman Comparison of in-cell methods In-cell NMR In-cell PELDOR In-cell spFRET Disturbance of native environment Yes Yes No Cell t>pc Toxicity Subcellular localization Tag requirement Measurement time span Structural information1 X. laevis egg/oocyte X. laevis egg/oocyte E. coli, mammalian ccllsa Sequence dependent15 Sequence dependent15 No Nucleus/cyto solc Nucleus/cytosolc Nuclcusd No Yes Yes Hours < 70 mine Hours Short-range Long-range Long-range in-cell NMR of nucleic acids in mammalian cells SLO-delivered dsDNA in leLa cells 14.0 13.5 13.0 12.5 12.0 ppm (R. Hansel and V. Dotsch - unpublished) In-cell Raman microscopy (mammalian cells): under development Raman Shift (cm"')