Nucleic Acids in Chemistry and Biology

The donor–acceptor distance (R) is related to ETby the system constant Ro, which is the distance at
which there would be 50% of maximal energy transfer [Equations (11.2) and (11.3)]:

(11.2)

(11.3)

From these relationships, FRET theory predicts that fluorescence transfer depends on the sixth power
of the distance between partner molecules. In practice, the distances over which FRET can be measured vary
between 40 and 100Å (Figure 11.3). There are several factors that can influence the extent of fluorescence
transfer, and hence precautions need to be taken in order to account for all possible energy transfer pathways
and to properly quantify FRET measurements.
There are a few naturally occurring fluorophores found in nucleic acids, for example wyosine found in
tRNA. More commonly synthetic fluorophores (e.g.Figure 8.13) can be introduced into oligonucleotides. Such
modifications are extremely useful for studying nucleic acid structure and function. For FRET experiments,
in general one or both of the donor and/or acceptor is either covalently or non-covalently attached to a
nucleic acid, often viachemical synthesis.
Site-specific labelling of longer (thousands of base pairs) DNA samples remains problematic, although
recent advances in the use of sequence-specific methyl transferases to add specific labels to defined DNA
sites are promising. Normally DNA methyl transferases (MTases) transfer an activated methyl group to
the N-6 position of an adenine or C-5/N-4 position of a cytosine within a specific cognate sequence. By
use of a fluorophore and a flexible linker, sequence-specific labelled DNA can be obtained in a methyl
transferase–catalysed reaction. This technique developed by Elmar Weinhold is known as SMILing DNA.
David Lilley and co-workers have used FRET to delineate the overall geometry of four-way DNA junc-
tions^3 and the fold of the hammerhead ribozyme.^4 In addition, FRET is a useful method for distinguishing
between intercalation and groove binding in small molecule–DNA interactions (Section 9.2), since in general
FRET is observed during intercalation but not during groove binding.

RR

E

E





o

T

T

1

16









/

E

R

T RR

o

o





6

66

430 Chapter 11

Figure 11.3 A typical relationship between fluorescence enhancement, resulting from FRET, and the distance between
donor and acceptor fluorophores

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