Chemistry Reference
In-Depth Information
3.2 Extra- and Intracellular Targeting of Biomolecules
The location and dynamics of biomolecules like proteins play an important role in
cell signal transduction. Similarly relevant are issues like the assessment of molec-
ular function of biomolecules, e.g., for cancer research and target quantification.
A prerequisite, e.g., for monitoring molecular function in vivo is the ability to track
biomolecules within their native environment, i.e., on the cell surface or inside
cells, and needs to be met by any fluorescent label suitable for this purpose. The
challenges here include intracellular delivery of the chromophore as well as selec-
tive labeling of the target biomolecule within its native setting without affecting its
function. The latter is the prerequisite for assessing changes in the local envi-
ronment or the distances between labeling sites using hetero-FRET (chemically
different chromophores) or homo-FRET (chemically identical chromophores).
Successful experiments require the selection of labels that are matched with the
biological system, for instance, the location of the target (cell surface, intracellular,
or vascular compartments), the expression level of the target, or whether the target
is within a reducing versus an oxidizing environment.
The report of several established and recent methods for extracellular and
intracellular labeling of biomolecules, in conjunction with some commercial tools
for these applications [
99
] is mainly advantageous for organic fluorophores. This
includes several strategies for site-specific covalent and noncovalent labeling of
biomolecules, typically proteins, in living cells. Examples are enzyme-catalyzed
labeling by posttranslational modification, as in biotin ligase-catalyzed introduction
of biotin into biotin acceptor peptides, which may be used to label proteins at the
cell surface. Both intracellular and surface labeling have also been achieved by
specific chelation of membrane-permeant fluorescent ligands (biarsenical dyes such
as FIAsH or ReAsH bind to the tetracysteine motif, Ni-nitriloacetic acid (NTA)
conjugates bind to the hexahistidine motif, and Zn conjugates), or by self-labeling,
in which proteins fused to O6-alkylguanine-DNA alkyltransferase are combined
with enzymatic substrate derivatives (O6-alkylguanine-DNA alkyltransferase
(AGT) or SNAP-tags) [
1
,
99
]. Other alternatives present the HaloTag technology,
exploiting a modified haloalkane dehalogenase designed to covalently bind to
synthetic ligands which can be used for the highly specific labeling of fusion
proteins in living or chemically fixed cells and irreversible capture of these proteins
onto solid supports [
100
] or the use of 2,4-diamino-5-(3,4,5-trimethoxybenzyl)
pyrimidine (trimethoprim or TMP). For organic labels, also several methods are
well established for fluorophore delivery into cells. This includes acetomethoxy-
methyl (AM) ester derivatization as well as simple microinjection, gene guns,
cationic liposomes, controlled cell volume or cell membrane manipulation, and
endocytosis [
101
] or electroporation [
102
]. In particular the first strategy which
renders the dyes cell permeable, presents a huge advantage for this class of labels.
Meanwhile, extracellular targeting with QDs has been frequently reported [
103
].
Moreover, strategies have been described to reduce nonspecific QD binding and
uptake as a prerequisite for applications, where specific cell-chromophore
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