Homonuclear assignments fornucleic acids

(2) Nonexchangeable protons

Nick Ulyanov

May 4, 2004

Examples

1. 34-nt RNA Du et al. 2004 Biochemistry, in press.

2. 20-nt RNA Comolli et al. 2002 Nucleic Acids Res. 30:4371-9.

3. 27-nt DNA Ulyanov et al. 2002 J. Biomol. NMR 22:265-80.

Why homonuclear assignments?

1. Sequential assignments are alwayshomonuclear for nucleic acids.

2. Assignments of NOESY cross-peaks

Atom notations for bases

Atom notations for sugars

IUPAC-IUB Joint Commission onBiochemical Nomenclature (JCBN)

Eur. J. Biochem. 131: 9-15(1983).

H1'-H2' correlations

34-nt RNA

DQF-COSY

25 deg.

H5-H6 and methyl-H6 correlations inDNA

27-nt DNA, TOCSY 30 ms, 10 deg.29-nt DNA, TOCSY 30 ms, 20 deg.

H5-H6 correlations in RNA

20-nt RNA, TOCSY 50 ms, 30 deg.

20-nt RNA

natural abundance 13C-HMQC

30 deg.

C1'-H1' correlations in RNA

20-nt RNA, natural abundance 13C-HMQC, 30 deg.

Other ribose correlations in RNA

20-nt RNA

natural abundance

13C-HMQC

30 deg.

H6/H8-H1' walk

RNA DNA

Intra INTER

DNA 3.6-3.9 3.0-4.8

RNA 3.4-3.9 4.4-5.2

DNA H6/H8-H1' walk

27-nt DNA, 150 ms NOESY, 10 deg.

A4

A5

T6

T7

RNA H6/H8-H1' walk

A4

G5

U6

C7

20-nt RNA, 400 ms NOESY, 30 deg.

HCN correlations

N1C6

H6C1'

H1'

H6 -> (C6) -> N1 H1' -> (C1') -> N1

34-nt RNA

200 ms NOESY

25 deg.

H6/H8-H2'/H2" walks

RNA

DNA

H2'-H6/H8 H2"-H6/H8

Intra INTER Intra INTER

DNA 2.0-3.6 2.2-4.0 3.4-4.5 2.0-3.4

RNA 3.4-4.3 1.8-2.6

DNA

DNA H6/H8-H2' walk

A4

A5

T6

T7

27-nt DNA, 150 ms NOESY, 10 deg.

RNA H6/H8-H2' walk

20-nt RNA, 400 ms NOESY, 30 deg.

A4

G5

U6

C7

H6/H8-H3' walk

RNA DNAIntra INTER

DNA 2.5-4.7 3.7-5.1

RNA 2.7-3.5 3.0-3.8

DNA H6/H8-H3' walk

A4

A5

T6

T7

27-nt DNA, 150 ms NOESY, 10 deg.

presat

RNA H6/H8-H3' walk

C14

A15

C16

G17

34-nt RNA, 200 ms NOESY, 25 deg.

H6/H8-H4' walk

RNA DNA

Intra INTER

DNA 4.3-5.2 5.6-6.4

RNA 3.9-4.8 5.3-6.2

27-nt DNA, 150 ms NOESY, 10 deg.

H6/H8-H5'

RNA DNA

H5'-H6/H8 H5"-H6/H8

Intra INTER Intra INTER

DNA 3.6-4.9 (6.9-7.8) 3.7-4.4 (6.6-7.4)

RNA 3.6-4.5 (6.8-7.4) 4.0-4.7 (5.9-6.5)

DNA

H6/H8-H5"

RNAγ = gauche+

Slices through base protons H6/H8

27-nt DNA

150 ms NOESY

10 deg.

34-nt RNA

200 ms NOESY

25 deg.

H1'-H2' walk

RNA DNA Intra INTER

DNA 2.7-3.0 (4.5-7.2)

RNA 2.7-2.8 3.9-4.4

H1'

H2'

RNA H1'-H2' walk

20-nt RNA

400 ms NOESY

30 deg.

Slice through H1'

27-nt DNA, 150 ms NOESY, 10 deg.

Aromatic region

27-nt DNA, 150 ms NOESY, 10 deg.

Anomeric region

27-nt DNA, 150 ms NOESY, 10 deg.

Base-to-methyl region in DNA

27-nt DNA, 150 ms NOESY, 10 deg.

Slice through methyl group

27-nt DNA, 150 ms NOESY, 10 deg.

H1'-to-Adenine H2

A5

T6

T24

T23

H1'-to-Adenine H2 region

27-nt DNA, 150 ms NOESY, 10 deg.

A5

T6

T24

Assignments of adenine H2 protons

1. Based on water NOESY

2. Based on T1 inversion recovery experiment

3. From C2-H2 correlations

20-nt RNA

natural abundance 13C-HMQC

30 deg.

27-nt DNA, 10 deg.

Some general advice…

1. Not having heteronuclear data at all is a badidea, especially for RNA.

2. Because sequential assignments areultimately based on a structure, take all yourassumptions with a grain of salt. Go back toassignments after you think you alreadyfinished the refinement.

3. Get a reasonable model of your molecule forassignments of NOESY cross-peaks, and use"assignment distances" ("ad") tool in Sparky.