Biomedical Engineering Reference
In-Depth Information
nucleobase C-H RDCs (C5-H6, C6-H5, C4-H5).
96
In general, the measure-
ment of such small RDCs is only practical for small-to-moderate size RNA
molecules
(,30
nt)
and
can
become
challenging
for
much
larger
RNA
molecules.
Experiments have also been developed to measure
1
H-
1
HRDCs.
107-110
For
example, Pardi and co-workers recently demonstrated the measurement of imino
1
H-
1
H RDCs in a 29-nt IRE RNA, and showed their utility in differentiating
geometric differences between GU and Watson-Crick (WC) base pairs.
109
Bax
and co-workers developed a CH
2
-S
3
E experiment to measure geminal H59-H599
RDCs on a 24-nt RNA. By incorporating the 'Rance-Kay' transfer element,
111
undesired magnetisation transfers were suppressed more than 10-fold, leading to
significantly narrowed
1
H linewidths and enabling the accurate measurement of
RDCs between these methylene pairs.
108
The authors note that RDC values are
negative in sign, similar to other sugar RDCs, and indicate that their orientation
is parallel with respect to the helical axis, as expected for a helical geometry.
108
In
another interesting application, experiments have been developed to detect and
measure longer range
1
H-
1
H RDCs between nuclei that are up to 12
˚
apart.
107
These experiments use selective decoupling pulses to suppress line broadening
contributions from
1
H-
1
H dipolar couplings and thereby permit the accurate
measurement of small (ca. 1 Hz) RDCs between the well-resolved sugar H19 and
nucleobase H5 nuclei.
107
Selective labelling strategies have also been used to help overcome the
spectral resolution problem in the measurement of RDCs. For example,
Lukavsky and co-workers were able to nearly double the number of RDCs
(compared to a uniformly
13
C/
15
N-labelled sample) for a 74-nt RNA by
ligating a uniformly
15
N-labelled strand to an unlabelled strand.
112
In another
interesting application, Luy and Marino incorporated
19
F into the sugar 29-
hydroxyl position of a 21-nt RNA at different sites and used these constructs
to measure F-H (F29-H29,F29-H19,F29-H39,F29-H6, F29-H8) RDCs. The
authors find that RDCs fit extremely well to an A-form geometry in helical
regions, indicating that this probe does not perturb the helical geometry.
113
Although not reviewed here, one can also measure a wide variety of residual
chemical shift anisotropies (RCSAs) as a complement to RDCs.
99,114,115
Sizeable RCSAs can be measured in nucleobase carbons and nitrogens as an
offset in the observed chemical shift following alignment of the RNA. Here,
care has to be taken to account for any changes in chemical shift arising from
interactions with the ordering medium.
114,116-118
Rather than report on the
orientation of the axially symmetric inter-nuclear bond vector relative to a
molecular frame, RCSAs report on the orientation of the typically asymmetric
chemical shift anisotropy (CSA) tensor centered at a given nucleus (typically
nucleobase
13
Cand
15
N and backbone
31
P).
87,114,118-120
Because the CSA of
protonated nucleobase carbons and nitrogens are often non-coincident with
the C-H and N-H bonds, RCSAs can provide independent orientation
information. Moreover, unlike axially symmetric RDCs, asymmetric RCSAs
are sensitive to rotations along the CSA principal direction, making them in
