Lecture 12 M230 Feigon Sequential resonance assignments in DNA (and RNA): homonuclear method 2 structure determination Reading resources Evans Chap 9 The wonderful world of NUCLEIC ACID NMR! Catalytically essential pseudoknot in human telomerase RNA Telomere repeat DNA quadruplex with bound and free cations Double-strand RNA binding domain of yeast RNase III in complex with RNA target
Why do NMR of nucleic acids? More than a simple double helix!! Unusual DNA structures triplexes quadruplexes bent DNA Drug-DNA complexes Chemically modified DNA, e.g. cis-t (anti-cancer drug) Cation localization rotein-dna complexes plus, the wonderful world of RNA! Ribozymes seudoknots viral RNAs snrnas snornas & scarnas Aptamers Riboswitches mirnas, sirnas Telomerase RNA lncrnas and lincrnas HIV RNA RNA-protein complexes Etc! Nucleic acids different problem than proteins! Only four bases, one sugar Simpler! Lower proton density nucleotide is 3X MW of aa For helices, no long range constraints No through-bond connectivity from nucleotide to nucleotide (except through ) More difficult!
1 H NMR of Nucleic Acids: Assignments 1) roton resonances Non-exchangeable DNA Base AH8, GH8 AH2 TH6, CH6 CH5, TMe Deoxyribose 1, 2, 2, 3, 4, 5, 5 RNA same, except UH5, UH6 (like CH5, CH6) ribose, no 2 (2 only) Exchangeable (only seen in H 2 O) iminos amino 2) roton spin systems - D2O only; no coupled labile spin systems (except aminos) CH5-CH6, UH5-UH6 ~7 Hz TMe-TH6 4 bond coupling ~1.5 Hz deoxyribose ribose aromatic 5, 5 HCH O base H3 H2 H4 H1 ~1.5 Hz H2 DNA deoxyribose 2 1 3 4 2 5 5 TMe-TH6 ~1.5 Hz (long range coupling) RNA: UH5-UH6 RNA ribose 1 2 3 4 5 5 ~7 Hz Minor groove: sugar, AH2, G amino Major groove: H8, TMe, CH5, A & C aminos Also, 31, proton attached 13 C and 15 N And 13 C- 1 H, 15 N- 1 H, 31-13 C, and 31-1 H J coupling CH5-CH6 ~7 Hz
3) 1D spectra of a DNA dodecamer with N 6 A mod. A. Non-exchangeable 1) intensities Me 3x aromatics 2) chemical shifts -- see regions on spectrum 3) coupling constants -- CH5-CH6 -- all sugar protons are coupled *! 1 2 3 4 5 6 7 8 9 10 11 12! C G C G A A T T C G C G! G C G C T T A A G C G C! 12 11 10 9 8 7 6 5 4 3 2 1! *! * = m 6! Exchangeable proton resonances: iminos and aminos *! 1 2 3 4 5 6 7 8 9 10 11 12! C G C G A A T T C G C G! G C G C T T A A G C G C! 12 11 10 9 8 7 6 5 4 3 2 1! *! * = m 6! Strange appearance of aromatic region [attenuated intensities] is due to water suppression (sample in 95% H 2 O). resence of imino resonances between 12-15 ppm indicates stable base pairing, important monitors of folded structure. Each Watson-Crick base pair gives rise to one imino resonance. Terminal iminos may be exchange broadened due to breathing. Imino region is free of protein resonances, good for monitoring complex formation with proteins and drugs.
Assignment of spin systems COSY -- can identify all CH5-CH6 TMe-TH6 H1 -H2, H2, etc. Theoretically, can isolate each deoxyribose spin system 1 2 2 3 4 5 5 but 5, 5 usually difficult or impossible due to overlap with 4 TOCSY -- connects whole spin system, so can resolve more xpeaks, e.g. 2, 2-5, 5 ; 1-3 aromatic H1, CH5 1 [d( C 2 G 3 C 4 G 5 A 6! *A 7 T 8 T 9 C 10 11 12! G C G )]2!
*! 1 2 3 4 5 6 7 8 9 10 11 12! C G C G A A T T C G C G! G C G C T T A A G C G C! 12 11 10 9 8 7 6 5 4 3 2 1! *! * = m 6! H1 H1, CH5 2, 2 Me aromatic 1) Base-sugar interactions Sequential Assignment of A- or B-DNA Helices! rimarily relies on NOESY spectra (next page) (vs NOESY and COSY for 1 H sequential assign. of proteins) [For DNA, most of COSY information is also in NOESY since coupled protons generally also show NOEs] The H6 (pyr) & H8 (pur) protons are close (within 4Å) to 1, 2, 2 protons on own base (intranucleotide NOE) and to 1, 2, 2, protons on 5 neighboring base (internucleotide NOE)! 3 5 base H1, 2, 2 base H1, 2, 2 base H1, 2, 2 Intensities of xpeaks will depend on sugar conformation B-DNA with anti glycosidic conformation closest to intra 2 inter 2 next page but also directly (and indirectly) to others. Can assign DNA sequentially by xpeaks from base to sugar to base... NOEs expected base - H1 base - H2, H2 2 each H1 - H2, H2 like COSY xpeaks
Sequential assignments of DNA (& RNA) A T A Sequential assignments of DNA (& RNA) A T A
H1 C 1 H6, H8 AH2 AH2 T7 C 1 C3 C11 T8 C9 G2, G12 G10 G4 A6 A5 *Note: This is two peaks. Can also do same thing via base - H2, H2 (as in schematic) but now have 4 xpeaks base more complicated. Useful to double-check assignments (and exclude AH2s). Assigned H1 can be connected to rest of sugar spin system via COSY, TOCSY, and/or NOESY. Have now assigned all H8, H6, H5, TMe, and sugar resonances (except 5, 5 usually). Note: NOEs to H1 usually only show up clearly at long τ m and are partially spin diffusion, so after assignments are made may want to use shorter τ m for structure determination. 2) The AH2 protons are usually far from any other protons and have few or no NOEs in D 2 O. Often (usu. weak) NOEs to H1 or AH2-AH2: these give information on local structure (minor groove width, propellering). Assignment to specific base is done via NOE from iminos in H 2 O. Also, 1 H- 13 C HSQC
3) Base-base interactions Most interactions in DNA are along strand, very few cross strand NOEs - pyr (CH5, TMe) - pur (H8) and pyr (H6) Directional - goes 3 5 only in N 6 MeA Dickerson dodecamer 1 2 3 4 5 6 7 8 9 10 11 12! C G C G A A T T C G C G! 5 See NOESY spectrum strong weaker T 8 Me-T 7 Me T 8 Me-T 7 H6 C 3 H5 -G 2 H8 T 7 Me-A 6 H8 C 11 H5-G 10 H8 C 9 H5 -T 8 H6 in base-h1 region Note that if both strands were not symmetrical, would have to assign along each strand. *! 1 2 3 4 5 6 7 8 9 10 11 12! C G C G A A T T C G C G! G C G C T T A A G C G C! 12 11 10 9 8 7 6 5 4 3 2 1! *! * = m 6! T7 T8 H1 H1, CH5 2, 2 Me aromatic fingerprint
DNA conformation 1) a) Appearance of sequential base-sugar NOEs and base-base interactions indicates a right handed strand conformation. b) Strong base-h2, H2 and weaker base-h1 NOEs indicates anti conformation of bases. c) Intensity of base-base and base-sugar NOEs indicative of local conformation s.a. base tilt, twist, sugar pucker, groove width, etc. 2) Since there are few xstrand NOEs, double strand relies on: a) appearance of H-bonded imino resonances in H 2 O b) melting studies c) direct detection of H-bonds via scalar coupling (requires labeled sample) ~ -88 Hz ~2.5 Hz 15N H 15 N ~6-10 Hz DNA conformation 3) Sugar conformation a) Measure coupling constants via DQF-COSY b) Deduce sugar conformation from NOE intensities 5, 5 HCH 4 3 2 2 1 C2 endo J 1, 2 ~ 10 Hz (S-type) B-DNA short distance intra nucleotide H6,H8 H2 inter nucleotide H6,H8 H2 5, 5 HCH 4 3 2 2 1 A-DNA (or RNA) intra nucleotide H6,H8-H3 ; H4 -H2 inter nucleotide H6,H8-H2 ; H6,H8-H3 C3 endo J 1, 2 ~ 1.5-3.3 Hz (N-type) If use short enough τ m, can distinguish.
Why is RNA harder to study than DNA? Overlap of ribose H2, H3, H4, H5, H5! C and U base protons not distinguishable by J-coupling or chemical shift (since no Me on U) aromatic H1, UH5 CH5 H2, H3, H4, H5, H5! Hairpin Ribozyme Loop B: An irregular underwound helix with 7 non-watson Crick base pairs A40 A23 U39! Butcher, Allain, Feigon, Nature Struct. Biol. 6, 212-216 (1999)!