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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.