reactions to functionalize [9–13] and image RNA in cells [14–
19]. Nevertheless, new chemoselective reactions that can be
tuned to work in aqueous and under milder conditions are constantly needed to expand the repertoire of functionalized RNA. In
this context, palladium (Pd)-catalyzed C-C bond formation, which
is a powerful reaction in synthetic organic chemistry, is proving
useful as a chemoselective transformation for post synthetic labeling of biomolecules [20–23].
Like other methods, Pd-mediated cross-coupling reactions
were first established on DNA and protein [20, 21]. Bromo- or
iodo-modified DNA oligonucleotides (ONs) and proteins,
prepared by chemical or enzymatic methods, are reacted with a
cognate reactive handle tagged with a reporter in the presence of
a water-soluble Pd-ligand catalytic system. The Manderville and
J€ aschke groups developed aqueous-phase Suzuki-Miyaura coupling
reaction to functionalize DNA by reacting halogen-modified
DNA ONs with boronic acid/ester substrates in the presence
of Pd(OAc) 2 and a water-soluble triphenylphosphane-3,3
0 ,
3
0 -trisulfonate ligand [24, 25]. However, this catalyst-ligand combination requires elevated temperature (>70
C), basic pH buffer
conditions, and long reaction times to produce coupled ON products. Understandably, RNA will not survive these high temperature and basic pH buffer conditions. Meanwhile, Davis and Lin
group formulated a Pd-ligand catalytic system to execute Suzuki
and Sonogashira reactions, respectively, on proteins under very
mild conditions [26, 27]. The catalytic system was made of
Pd(OAc) 2 and 2-aminopyrimidine-4,6-diol (ADHP) or
dimethylamino-substituted ADHP (DMADHP), which enabled
the direct incorporation of functional labels onto DNA ONs at
room temperature and at a pH (8.5) slightly higher than physiological pH [28]. Encouraged by this report, we established a milder
and efficient Suzuki-based labeling method to generate RNA functionalized with different biophysical reporters [29]. In this strategy,
5-iodouridine-5
0 -triphosphate (IUTP 1) was incorporated into
RNA transcripts by in vitro transcription reaction catalyzed by T7
RNA polymerase. Iodo-labeled transcripts were then posttranscriptionally reacted with various cognate reactive partners (boronic
acid/ester substrates) labeled with a desired biophysical reporter
or tag in the presence of Pd(OAc) 2 and ADHP or DMADHP
complex. The reaction proceeds under benign conditions (37
C
and pH 8.5) and produces coupled RNA products in moderate to
good yields. Taken together, this method is modular, and provides
access to RNA labeled with (a) fluorogenic environment-sensitive
nucleoside for probing nucleic acid structure and recognition,
(b) fluorescent probes for microscopy, and (c) affinity tags for
pull-down and immunoassays (Fig. 1). Further, metabolic labeling
474
Manisha B. Walunj and Seergazhi G. Srivatsan
19]. Nevertheless, new chemoselective reactions that can be
tuned to work in aqueous and under milder conditions are constantly needed to expand the repertoire of functionalized RNA. In
this context, palladium (Pd)-catalyzed C-C bond formation, which
is a powerful reaction in synthetic organic chemistry, is proving
useful as a chemoselective transformation for post synthetic labeling of biomolecules [20–23].
Like other methods, Pd-mediated cross-coupling reactions
were first established on DNA and protein [20, 21]. Bromo- or
iodo-modified DNA oligonucleotides (ONs) and proteins,
prepared by chemical or enzymatic methods, are reacted with a
cognate reactive handle tagged with a reporter in the presence of
a water-soluble Pd-ligand catalytic system. The Manderville and
J€ aschke groups developed aqueous-phase Suzuki-Miyaura coupling
reaction to functionalize DNA by reacting halogen-modified
DNA ONs with boronic acid/ester substrates in the presence
of Pd(OAc) 2 and a water-soluble triphenylphosphane-3,3
0 ,
3
0 -trisulfonate ligand [24, 25]. However, this catalyst-ligand combination requires elevated temperature (>70
C), basic pH buffer
conditions, and long reaction times to produce coupled ON products. Understandably, RNA will not survive these high temperature and basic pH buffer conditions. Meanwhile, Davis and Lin
group formulated a Pd-ligand catalytic system to execute Suzuki
and Sonogashira reactions, respectively, on proteins under very
mild conditions [26, 27]. The catalytic system was made of
Pd(OAc) 2 and 2-aminopyrimidine-4,6-diol (ADHP) or
dimethylamino-substituted ADHP (DMADHP), which enabled
the direct incorporation of functional labels onto DNA ONs at
room temperature and at a pH (8.5) slightly higher than physiological pH [28]. Encouraged by this report, we established a milder
and efficient Suzuki-based labeling method to generate RNA functionalized with different biophysical reporters [29]. In this strategy,
5-iodouridine-5
0 -triphosphate (IUTP 1) was incorporated into
RNA transcripts by in vitro transcription reaction catalyzed by T7
RNA polymerase. Iodo-labeled transcripts were then posttranscriptionally reacted with various cognate reactive partners (boronic
acid/ester substrates) labeled with a desired biophysical reporter
or tag in the presence of Pd(OAc) 2 and ADHP or DMADHP
complex. The reaction proceeds under benign conditions (37
C
and pH 8.5) and produces coupled RNA products in moderate to
good yields. Taken together, this method is modular, and provides
access to RNA labeled with (a) fluorogenic environment-sensitive
nucleoside for probing nucleic acid structure and recognition,
(b) fluorescent probes for microscopy, and (c) affinity tags for
pull-down and immunoassays (Fig. 1). Further, metabolic labeling
474
Manisha B. Walunj and Seergazhi G. Srivatsan