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Heteronuclear NMR of Nucleic Acids
In most cases, requires samples isotopicaly enriched
by 13C and 15N (except for HSQC, HMQC)
Assignment uses NOE or through-bond experiments
Traditional constraints (NOEs, J-couplings)
Novel constraints (RDCs, residual CSA)
Studies of intramolecular dynamics

RNA preparation
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Labeled NTPs
Available commercially
Can be grown in cells
E. Coli
13C-glucose, 15N-ammonium
M. Methylotropus
13C-methanol, 15N-ammonia

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Assignment procedure for labeled NA
NOE based (I)

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Assignment procedure for labeled NA
NOE based (II)

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Assignment procedure for labeled NA
Through bond correlations (I)

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Assignment procedure for labeled NA
Through bond correlations (II)

Correlation of exchangeable protons with 15N
> > > > > > > > > > > > > > > > > >
Gradient sensitivity-enhanced HSQC
Kay, Keifer, Saarinen, JACS 1992.
A
U
C
G
G
C
G
C
C
G
U
U
C
G
1
5
10

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Correlation of exchangeable and non-exchangeable protons
HCCNH-TOCSY, Fiala et al. JACS 1996, Sklenar et al. J. Biomol. NMR 1996.

Correlation across the hydrogen bond
HNN-COSY experiment
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Dingley and Grzesiek, JACS 1998

Identification of hydrogen and carbon atoms in bases and sugars
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Constant-time 1H-13C HSQC experiment
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base
sugar

Assignment of non-exchangeable protons:
HCCH-type experiments


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Sugar to base correlation – the HCN experiment
Sklenar et al., J. Biomol. NMR 1993, 1994, Fiala et al., J. Biomol. NMR 1998, 2000.

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Sugar to phosphate correlation – the HCP experiment


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Dipolar couplings
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•Dipolar couplings add to J couplings
•They show up as a field or alignment   media dependence of the coupling
•If the overall orientation of the molecule is known the orientation of the vectors can be
determined