**NMR RELAXATION STUDIES OF FAST INTERNAL MOTIONS IN
NUCLEIC ACIDS**

Lukáš
Trantírek, Erik Caha and __Radovan Fiala__

Investigations of intramolecular dynamics on the
nanosecond to picosecond time scale by solution NMR spectroscopy are based on
the spin relaxation properties of nuclei such as ^{15}N or ^{13}C.
The most commonly measured quantities are the longitudinal relaxation rate
constant, R_{1}, the transverse relaxation rate constant, R_{2},
and the steady state nuclear Overhauser effect (NOE). The values of these
experimentally obtained parameters can be expressed as linear combinations of
spectral density functions. The spectral density function characterizes the
overall rotational diffusion of the molecule as well as its intramolecular
motions. Two approaches exist for the interpretation of the experimental data.
In spectral density mapping, the values of the spectral density function J(ω)
at
characteristic frequencies are determined from the relaxation data [1]. The
"model-free" formalism assumes a particular form of the spectral
density function, whose parameters then characterize amplitudes and time scales
of the molecular motions [2].

The methods for measuring ^{15}N relaxation
parameters have been well established thanks to a large body of literature on
protein dynamics studied through amide nitrogen relaxation. Because of the
similarity of the spin environments, the procedures developed for protein amide
nitrogen are directly applicable to imino nitrogen of guanine and uracil [3].
However, ^{15}N relaxation study of nucleic acids can yield the dynamic
properties of guanine and uracil bases only. For a more complete picture, the
use of ^{13}C relaxation data is highly desirable. The most suitable
candidates for relaxation studies in nucleic acids are C8 carbons of purines,
C6 carbons of pyrimidines and C1' of the sugar.

In order to bring insight into the internal dynamics
of RNA tetraloops we have performed a ^{13}C NMR relaxation and
molecular dynamics study of 14-nt RNA hairpins GGCAC__UUCG__GUGCC and GGCAC__GCAA__GUGCC
(the underlined nucleotides form the loops). The UNCG and GNRA families of
stable RNA hairpins (where N is any nucleotide and R is purine) have very
similar overall folds. However, the biological roles of these two sequences
appear different. The differences have been attributed to distinct dynamical
properties of the two sequences [4].

We have measured R_{1} and R_{1ρ}
relaxation rates for C8 of purines, C2 of adenines, C6 and C5 of pyrimidines as
well as for C1' of the ribose sugars at several magnetic field strengths. The
data have been interpreted in the framework of modelfree analysis
characterizing the internal dynamics of the molecules by order parameters and
correlation times for fast motions on the picosecond to nanosecond time scale
and by contributions of chemical exchange.

While both tetraloops exhibit increased mobility on
the fast time scales, with the GCAA loop we have detected a significant
contribution of conformational dynamics on the millisecond to microsecond time
scale. This is consistent with the observations that the GNRA family appears
more flexible and tolerant of the conformational changes important for
molecular recognition.

[1] G. Lipari & A. Szabo, *J. Amer. Chem. Soc.*
104 (1982) 4546-4559.

[2] J. W. Peng & G. Wagner, J. Magn. Reson. 82
(1992) 308-332.

[3] M. Akke, R. Fiala, F. Jiang, D. Patel & A. G. Palmer, *RNA,* 3
(1997) 702-709.

[4]
G. Varani, *Annu. Rev. Biophys. Biomol. Struct*., 24 (1995) 379-404.