Fluorescence Technologies for Monitoring Interactions Between Biological Molecules In Vitro - Progress in Molecular Biology and Translational Science - page 126

plasmid DNA, siRNA, and single-stranded antisense molecule). 50 The main
development of CPPs has focused on noncovalent delivery involving
formation of mixed complexes between peptides and nucleic acids, 51,52
which improve the cellular internalization of oligonucleotides. 50,53 In
this case, the ability of peptides to form stable interactions with nucleic
acids constitutes a key parameter for the selection of potent carriers, and
both extrinsic and intrinsic fluorescence have been used for screening
peptides and for a better understanding of peptide/nucleic acid complex
formation.
Analysis of peptide/nucleic acid interactions can be carried out by
monitoring intrinsic peptide fluorescence as well as specific labeling of
the cargo. It has been demonstrated for several CPPs that the fluorescence
emission maximum of their tryptophan residues is affected by the presence
of increasing amounts of nucleic acids and that the fluorescence emission
maximum of a labeled oligonucleotide varies with increasing concentra-
tions of the carrier peptide. Thus by monitoring both the intrinsic trypto-
phan fluorescence of peptides and the extrinsic fluorescence of a labeled
cargo, it is possible to investigate the stability of CPP/nucleic acid interac-
tions and thereby characterize complex formation. 54-57 The intrinsic
fluorescence of tryptophans within peptides usually exhibits a strong
quenching in the presence of nucleic acids. 54,55,58,59 This quenching
might be attributed to both direct peptide/nucleic acid interactions and
peptide/peptide interactions that occur when forming complexes with
nucleic acids. Although positively charged residues of peptide are able to
carry out electrostatic interactions with negative charges of the
phosphate groups of nucleic acids, changes in tryptophan fluorescence
intensity also suggest aromatic stacking effects. It has been demonstrated
that short peptides or protein domains are able to undergo tryptophan
fluorescence quenching when interacting with single-stranded
nucleic acids as well as DNA duplex and triplex. 60-63 For example,
strong binding of the KWGK peptide to a 21-mer duplex involves
intercalation and stacking interactions of the tryptophan with GC
regions of the oligonucleotide. 62 The quenching of fluorescence of the
tryptophan has been ascribed to an electron transfer from indole of the
tryptophan side chain (in the excited state) to purine and pyrimidine
bases. 62
With regard to extrinsic fluorescence of nucleic acids, the use of different
fluorescent probes can show distinct behaviors with peptides. Although
most of

the peptide/nucleic acid interactions induce fluorescence