Základy molekulární biofyziky (in English) Part 6: Cellular Structural Biology - NA DNA as a drug target Multiple x Multiple Single gene Multiple copies of mRNA copies of protein 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 (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" Tra n sc r i iption S Promoter Gene 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 X-ray NMR Na antiparallel basket parallel propeller hybrid-1 hybrid-2 2-tetrad antiparallel basket Wang et al. Structure (1993) 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 Drffracfion 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 if Salt Tube a Buffer 0 Tube a Precipitant ■ 1. 0.02 M Calcium chloride dihydrate ff 1. 0.1 M Sodium acetate tnhydrate pH 4.6 tr 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% v/v (+/-)-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% v/v 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% v/v Polyethylene glycol 400 X-ray diffraction ... underlying assumptions □ Lowest energy structure is biologically active Coordinates □ structure is independent of environmental conditions additives, hydration levels, temperature, MC, viscosity, concentration, ion type, ion strength,...... PDB statistics Exp. Method Proteins Nucleic Acids Protein-NA complexes Other X-ray 75 215 1 464 3 888 2 NMR | 8 737 1 030 | PI92 7 El. Microsc. A'M 128 0 Hybrid 46 3 2 1 Other 148 ■ 113 | 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) 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 b 1 37- 80- 38- 82-84- t i 39-40- 86- 41- 88- J 42- CI 7C4' 43- 15N ppm 145 8.5 8.0 7.5 7.0 1HPPm 150 -1- 5.0 4.0 6 5 4 3 d e 90- 0 g 95- § fid ^ 0 A 100 V imino amino -1-1- 13.5 13.0 12.5 8.0 7.5 7.0 6.5 ppm In-cell NMR: NA degradation (un)expected problem in cell in vitro ~ 5 hrs ~ 19 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 NAAR: 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™ ln-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 12.0 11.0 ppm Resolution limits the analysis of the polymorphs: site-specific labeles Crude cellular GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTA 13 12 11 10 pprrn a good news: DNA/RNA (if there is any) can be recovered from cells 12.0 11.0 ppm 12.0 11.0 ppm I cleared 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: • de 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 •Hlppm] •Hlppm] YES Spectral fingerprints in vitro - in vivo \^ YES Solve in vitro structure \ / N NO t Mimicking in vitro conditions? NO Solve in vivo structure Spectral fingerprinting: example Crude cellular homogenate Spectral fingerprinting: example Crude cellular homogenate 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 G2 arrested meiosis I meiotic meiosis II oocyte metophose interphase metaphase Cells accommodate/tolerate introduced NA Towards structural biology under native conditions X-ray NMR ln-cell NMR ln-cell spFRET In vivo 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 cell Ity i3C/i5N tyy fluorophorei_|> *\ paramag. label %■ 2H In-cell NMR ln-cell spFRET In-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 type Toxicity Subcellular localization Tag requirement Measurement time span Structural information* X. laevis egg/oocyte X. laevis egg/oocyte E. coli, mammalian cells3 Sequence dependent1* Sequence dependent No Nucleus/cytosoF Nucleus/cytosoF Nucleusd 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 HeLa 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 (cm1)