Chemistry Reference
In-Depth Information
protecting, caging, group allows the movement of mRNA to be studied at a higher
resolution. The photolytic unmasking of the
uorochrome, uncaging, activates the
fluorescent probes only at the illuminated region of the nucleus. This distinguishes
hybridized probes from the free oligo-dT, because the latter diffuse faster and
therefore disappear by dilution in the cellular volume.
An additional tool to label endogenous RNAs based on oligonucleotide hybridiza-
tion is provided by molecular beacons [8]. These molecules are characterized by a
particular stem
uorophoreand itsquencherbound
close together at each end of the probe. The aim of this system is to overcome the
background signal derived fromunbound probes, since the
loopstructure thatmaintains the
-
fluorescent signal will be
visible only when the annealing of the molecular beacon to its target separates the
quencher fromthe
uorophore.However, the stem
-
loop structure couldbe destroyed
in vivo by nuclease activity or protein binding, enabling
fluorescence without hybrid-
ization. Therefore, an improvement of this tool has been developed exploiting the
advantages of Fluorescence Resonance Energy Transfer (FRET, [9
-
11]). Brie
y FRET
occurs when two spectrally-matched
fluorescent pairs are suf
ciently close (
10 nm)
and in the correct orientation. The
fluorophore excited (donor) by an external source
(lamp) does not disperse all the energy in the emission instead it transfers activation
energy to the second longer-wavelength
fluorophore (acceptor) that in turn will emit.
With this technique, opportunely designed pairs of molecular beacons anneal to
adjacent sequencesonthesameRNAtarget, thus recruiting thedonorand theacceptor
of the FRET pair close enough to generate the FRET signal [12].
Another method to resolve the background due to unbound probes is the direct in
vitro labeling of the target RNA before introduction into the cell [13, 14]. In this case,
unlike previous techniques, the target RNA is not endogenously produced possibly
eliminating some steps in the normalmaturation pathway. SincemRNA injected into
the cytoplasmwill not have contact with the nuclear environment it could assemble a
differentmRNP complex. For example, mRNAs injected in the cytoplasmmay lack all
the nuclear factors usually recruited during their travel in this compartment.
Furthermore, even if injected into the nucleus, they may be de
cient in all the
factors deposited during transcription and maturation; processes like splicing and
polyadenylation. Nonetheless, these features are not always a con and they have been
exploited, for instance, to determine the involvement of nurse cell factors in bicoid
RNA dynamics in Drosophila embryos [15].
A completely different approach relied on the power of
fluorescently tagged
proteins [16
-
18]. Green Fluorescent Protein (GFP) and other
fluorescent proteins
derived from the jelly
sh Aequorea victoria are extensively used to tag RNA binding
proteins. In this case, the binding of the chimeric protein will indirectly label the
transcript. GFP-poly(A) binding protein 2 (GFP-PABP2) and the GFP-TBP export
factor [19] were used to study the movement of the bulk of endogenous mRNA by
Fluorescent Recovery after photobleaching (FRAP) experiments [20]. General RNA
binding proteins, like FIVH with oligo-dT, can be used to address endogenous
mRNAs but they do not discriminate one transcript from another, showing the
dynamics of a population and not of a speci
c transcript. Furthermore, there is the
additional complexity of the off- and on-rates of the protein and its recycling to
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