Biomedical Engineering Reference
In-Depth Information
Figure 9.3
Commonly measured RDCs in nucleobase and sugar moieties using pulse
sequences listed in Table 9.2. (A) One-bond C-H and N-H RDCs are
most often measured due to their favorable magnitude, but smaller one-
bond C-C and C-N as well as (B) two- and three-bond RDCs can be
measured. Note that typically larger magnitude RDCs are shown as thick
lines whereas smaller RDCs are shown as thin lines (see legend).
This can be achieved either by encoding individual components of the doublets
along the
1
H dimension,
71,98,99
or through intensity-based measurements in
TROSY-HSQC spectra with variable dephasing delays.
97
For example, by
selecting the TROSY component, Bax and co-workers demonstrated the
accurate measurement of C2H2, C5H5, C6H6, and C8H8 RDCs in the 60 nt
MMLV dimer initiation site RNA.
99
Multi-dimensional experiments that
employ HCN and E.COSY-type schemes can also be used to improve spectral
resolution, particularly for sugar C19 and nucleobase carbons and nitro-
gens.
96,100-103
In general, measurement of C-H RDCs in sugar moieties (e.g.,C29H29,
C39H39,C29H39,C39H29,C49H49,C59H59,C59H599) is significantly more
challenging because of severe spectral overlap in 2D C-H HSQC-type
experiments. Experiments have been developed that exploit the better
C19H19 resolution in measuring C29H29 and C39H39 RDCs.
104
Severe spectral
overlap unfortunately also complicates the measurement of RDCs between
31
P
and sugar protons
105,106
which can provide unique information on backbone
conformation,
a
problem
that
is
compounded
by
the
deterioration
of
sensitivity at high magnetic fields due to sizeable
31
P CSA relaxation.
Pulse sequences have also been developed to allow the precise measurement
of much smaller RDCs.
96,103,107-109
For example, Bax and co-workers
developed 3D HCN-type experiments for the measurement of very small
(22to+3 Hz) C-N RDCs (C19-N1/9, C6/8-N1/9, C2/4-N1/9) in a 24 nt RNA
and demonstrated the utility of these RDCs in determining small deviations
from idealised A-form geometry.
103
Experiments that rely on the planarity and
strong coupling between C6H6 and C5H5 bond vectors in pyrimidine
nucleobases have also been developed for the measurement of two-bond
