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!!

triplexes Unusual DNA structures quadruplexes

bent DNA

Drug-DNA complexes Chemically modified DNA, e.g. cis-Pt (anti-cancer drug) Cation localization Protein-DNA complexes

plus, the wonderful world of RNA! Ribozymes Pseudoknots 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 P) More difficult!

1H NMR of Nucleic Acids: Assignments

1) Proton 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 H2O) iminos amino

2) Proton 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

CH5-CH6 ~7 Hz

~7 Hz

~1.5 Hz

Minor groove: sugar, AH2, G amino Major groove: H8, TMe, CH5, A & C aminos

TMe-TH6 ~1.5 Hz 1’ 3’ — 4’ 2’

2’’

—

5’

5’’

—

5’

5’’ 1’ —2’ — 3’ — 4’

—

DNA deoxyribose

RNA ribose

O

H3’ H2’

base

H1’

H2’’

H4’

5’, 5’’ HCH

P

P

(long range coupling)

Also, 31P, proton attached 13C and 15N And 13C-1H, 15N-1H, 31P-13C, and 31P-1H J coupling

RNA: UH5-UH6

3) 1D spectra of a DNA dodecamer with N6A 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!

*!*! * = m6!

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!

*!*! * = m6!

Exchangeable proton resonances: iminos and aminos

 Presence 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.

Strange appearance of aromatic region [attenuated intensities] is due to water suppression (sample in 95% H2O).

Assignment of spin systems COSY -- can identify all

CH5-CH6 TMe-TH6 H1’-H2’, H2’’, etc.

Theoretically, can isolate each deoxyribose spin system

but 5’, 5’’ usually difficult or impossible due to overlap with 4’

1 2 3 4 5 6 7 8 9 10 11 12 ![d( C G C G A A T T C G C G )]2!*!

1’ 3’ 4’ 2’ 5’

2’’ 5’’

TOCSY -- connects whole spin system, so can resolve more xpeaks, e.g. 2’, 2’’ - 5’, 5’’ ; 1’-3’

aromatic H1’, CH5

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!

*!*! * = m6!

arom

atic

H1’, CH5 2’, 2’’ Me

H1’

⇒ Primarily relies on NOESY spectra (vs NOESY and COSY for 1H sequential assign. of proteins)

[For DNA, most of COSY information is also in NOESY since coupled protons generally also show NOEs]

Intensities of xpeaks will depend on sugar conformation base H1’, 2’, 2’’

base H1’, 2’, 2’’

base H1’, 2’, 2’’

3’

5’ B-DNA with anti glycosidic conformation closest to intra 2’

inter 2’’ but also directly (and indirectly) to others.

⇒ next page

∴ 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

(next page)

Sequential Assignment of A- or B-DNA Helices!

1) Base-sugar interactions 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)!

Sequential assignments of DNA (& RNA)

A

A

T

Sequential assignments of DNA (& RNA)

A

A

T

H1’

H6,

H8

*Note: This is two peaks.

T7 AH2

C3 C11 T8

C9 AH2

G4 G10 G2, G12

A6 A5

C1 ↓

C1 →

  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 τm and are partially spin diffusion, so after assignments are made may want to use shorter τm for structure determination.

2) The AH2 protons are usually far from any other protons and have few or no NOEs in D2O. 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 H2O. Also, 1H-13C 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 N6MeA Dickerson dodecamer

See NOESY spectrum strong weaker

1 2 3 4 5 6 7 8 9 10 11 12 !C G C G A A T T C G C G!

T8Me-T7Me T8Me-T7H6 C3H5 -G2H8 T7Me-A6H8 C11H5-G10H8 C9H5 -T8H6

in base-H1’ region

Note that if both strands were not symmetrical, would have to assign along each strand.

5’

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!

*!*! * = m6!

arom

atic

H1’, CH5

⇑ “fingerprint”

2’, 2’’ Me

H1’

T7 T8

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 H2O b) melting studies c) direct detection of H-bonds via scalar coupling (requires labeled sample)

15N H 15N

~ -88 Hz ~2.5 Hz

~6-10 Hz

DNA conformation 3) Sugar conformation a) Measure coupling constants via DQF-COSY b) Deduce sugar conformation from NOE intensities

B-DNA short distance intra nucleotide H6,H8 → H2’ inter nucleotide H6,H8 → H2’’

A-DNA (or RNA) intra nucleotide H6,H8-H3’; H4’-H2” inter nucleotide H6,H8-H2’; H6,H8-H3’

If use short enough τm, can distinguish.

C2’ endo J1’, 2’ ~ 10 Hz

C3’ endo J1’, 2’ ~ 1.5-3.3 Hz

2’

2’

1’

1’

2’’ 3’

P

4’

5’, 5’’ HCH P

2’’

3’

P

4’

5’, 5’’ HCH P

(S-type)

(N-type)

Why is RNA harder to study than DNA?

aromatic H1’, UH5 CH5

H2’, H3’, H4’, H5’, H5’’ !

 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)

Butcher, Allain, Feigon, Nature Struct. Biol. 6, 212-216 (1999)!

U39!

Hairpin Ribozyme Loop B: An irregular underwound helix with 7 non-Watson Crick base pairs

A40

A23