Structural Analyses of the Ribosome by Chemical Modification Methods - Biophysical Approaches to Translational Control of Gene Expression - page 74

are dangerous to work with and require special precautions. Additionally, DEPC
cannot be used with buffers containing Tris or HEPES, as the presence of large
numbers of amino groups in these reagents serves as a sink for the chemical probe.
These problems aside, radioactively labeled primer extension was an extremely
useful tool for many years. However, as discussed next, a number of technological
advancements have enabled researchers to overcome many of these limitations,
adding significant power and cost savings to chemical approaches to probing RNA
structure.

4.4 Modern Techniques

4.4.1 Introduction

Since 1998 a host of new technologies to circumvent the existing limitations of
chemical probing have been devised. The first of these technologies, in-line cleav-
age or in-line probing, involves backbone-based breakage of the RNA. This tech-
nique uses either spontaneous cleavage (Soukup and Breaker 1999 ) or uranyl-based
photocleavage (Wittberger et al. 2000 ) to locate breakages in the RNA. These are
followed by the same reverse primer extension as before. While useful for locating
certain points of weakness in the RNA structure, they tend to be most useful on
small molecules.

4.4.2 SHAPE (Selective 2 ¢ -Hydroxyl Acylation Analyzed by

Primer Extension)

In 2000 a new variant of chemical probing was developed (Chamberlin and Weeks
2000 ). This group created RNAs with a special 2¢ amine substitution and reacted
them with activated esters. Bases that had the 2¢ amine substitution in single-stranded
regions reacted faster than those in duplex or other base-pairing situations. They
found that this reactivity was not due to electrostatics or solvent accessibility, but
rather to local nucleotide flexibility. While an interesting finding, it was constrained
by having to create synthetic RNAs with this substitution. However, in 2005, a fur-
ther breakthrough was developed (Merino et al. 2005 ). Rather than having to create
synthetic RNAs, a chemical ( N -methyl isatoic anhydride [NMIA]) is used to modify
fl exible 2 ¢ hydroxyl groups in the sugar backbone of RNAs. Since this is a con-
served feature in every base, it allows the simultaneous interrogation of all bases in
a given molecule, identifying those bases with the greatest flexibility, i.e., those not
involved in base-pairing or other intermolecular interactions. The chemically
modified bases are subsequently identified by their ability to promote strong stops
in primer extension reactions. This represented a huge step forward for chemical
modi fi cation of RNAs for structural analysis .