An RNA spin-labeling technique was developed using the well-characterized interaction between the HIV Rev peptide and the Rev response element (RRE) RNA as a model system. Spin-labeled RNA molecules were prepared by incorporating guanosine monophosphorothioate (GMPS) at the 5′ end using T7 RNA polymerase and then covalently attaching a thiol-specific nitroxide spin label. Three different constructs of the RRE RNA were made by strategically displacing the 5′ end within the native three-dimensional structure. Nitroxide-to-nitroxide distance measurements were made between the specifically bound RNA and peptide using electron paramagnetic resonance (EPR) spectroscopy. The dipolar EPR method can reliably measure distances up to 25 Å, the calculation of which is derived from the 1/r3 dependence of the broadening of EPR lines in motionally frozen samples. This RNA-labeling technique, dubbed 5′ displacement spin labeling, extends the usefulness of the dipolar EPR method developed for analysis of protein structure. The advantage of this technique is that it is applicable to large RNA systems such as the ribosome, which are difficult to study by other structural methods.

A linear DNA molecule may be labelled at a fixed locus by a minute complementary radioactive molecule. A collection of identical molecules is to be so labelled and each one independently cut at a random number, N, of random places, X (N Poisson, X uniform). Fragments containing the label are to be collected and assayed by length. It is shown that the recovery pattern (fragment length distribution) contains a jump discontinuity at the fixed locus and may be used to determine the distance between the attachment site and the nearest end of the molecule.

The recovery pattern under the hypothesis that the collection of molecules are circularly permuted, i.e. the labelled locus is uniformly distributed over the length of the molecule, does not contain such discontinuities. The case where the labelling molecule has a non-negligible extent is also treated.