2.3 Analysing nucleic acid structures
In studying nucleic acid structures, many different experimental approaches can be adopted. In many cases, nucleic acid structures are examined in vitro, under non-physiological conditions, such as after denaturation or chemical synthesis. Nucleic acids within a cell are formed under very specific conditions and the structures that they adopt are influenced not only by the nature of their synthesis (by DNA or RNA polymerases), but by ancillary proteins that influence their folding. Nevertheless, there are a number of techniques that are routinely used to analyse nucleic acids in vitro. These techniques are described in Subsection 2.4. For example, electrophoresis not only gives us information about the length of a polynucleotide chain but it also provides some limited information about its shape, because the speed with which a polynucleotide moves through a gel matrix is related to the degree of compaction of the molecule.
Despite the success of resolving the structure of DNA by X-ray diffraction in the 1950s, the application of this technique to RNA was initially limited. There are two main reasons for this limitation. The first is that cells contain mixtures of RNA molecules and RNA is relatively unstable. Therefore, unlike for DNA, it is virtually impossible to purify individual species of RNA. Secondly, unlike DNA, for which a double-helical secondary structure is sufficient for most of its functions, RNA function, is critically dependent upon its tertiary structure. Therefore, like proteins, each RNA species must be isolated in sufficient quantities in its native form for successful crystallisation and analysis. Only relatively recently have X-ray diffraction studies made inroads into revealing the structures of RNA molecules, as we will see later in this unit, where we discuss the structure of ribosomal RNAs in relation to translation.