A modified guanosine phosphoramidite for click functionalization of RNA on the sugar edge

Article abstract of DOI:10.1039/c2cc34015a

A propargyl containing guanosine phosphoramidite was synthesized and incorporated into siRNA, enabling click-ligation with an azido fluorophore onto the nucleobase sugar edge. Duplex stability was not affected by labeling at this new site, which allowed deconvolution of the effects of label, structure and attachment site on RNAi activity.

Full text of DOI:10.1039/c2cc34015a

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A modified guanosine phosphoramidite for click functionalization of
RNA on the sugar edgewzy
Salifu Seidu-Larry, Bettina Krieg, Markus Hirsch, Mark Helm and Olwen Domingo*
Received 5th June 2012, Accepted 17th September 2012
DOI: 10.1039/c2cc34015a
A propargyl containing guanosine phosphoramidite was syn-
thesized and incorporated into siRNA, enabling click-ligation
with an azido fluorophore onto the nucleobase sugar edge.
Duplex stability was not affected by labeling at this new site,
which allowed deconvolution of the effects of label, structure and
attachment site on RNAi activity.
methylation of the exocyclic N2 amine of guanosine on base
pairing properties.
Altogether, this modification seems to have only a minimal
impact and a significant difference is first observed with double
methylation on position N2.7,12–14
We therefore decided to explore labeling of RNA at the
guanosine sugar edge in an siRNA model system. Research
associated with RNA interference,15 a process that moderates
gene expression in cells, is often accompanied by fluorescent
labeling of the siRNAs. This approach makes the intracellular
tracing of the siRNAs possible.16 The 50-end of the antisense
strand is a particular target for positional labeling on the sugar
edge. Because the 5’OH is phosphorylated in vivo, methyl-
ation of this OH-group has been reported to ablate RNAi
activity.17,18
To investigate in detail the effect of 50-siRNA labeling, we
designed and synthesized a phosphoramidite building block
for RNA synthesis, bearing a moiety whose alkyne functional
group would enable CuAAC-type labeling with an azide,
a method that has been used to great effect in bioconjugation
chemistry between fluorescent dyes and RNA/DNA.21–27
Here, the exocyclic N2 of guanosine was our target for a
diazotization reaction, followed by a nucleophilic substitution
by a fluoride ion.14,19,20,28
The labeling of biopolymers such as RNA is of out-
standing importance for investigations into structure–function
relationships.1 The attachment of fluorescent labels may,
however, influence both structure and function of the RNA,
depending on whether it involves the Watson–Crick, the
Hoogsteen or the sugar edge of a given nucleoside. In the
case of simple duplexes the secondary structure is essentially
restricted to the Watson–Crick edge. Hence, dye labeling
directly onto the Watson–Crick face of a given nucleobase is
expected to be detrimental to duplex formation. On the other
hand, popular labeling strategies on the Hoogsteen edge focus
mainly on the N7 or C8 of purines2–4 or the C5 position of
pyrimidines5,6 and although labeling directly onto the sugar
edge is a promising approach, such attachment points are so
far restricted to the 20-position of the ribose.7,8 Such deriva-
tization is, however, known to heavily affect the sugar pucker
and thus also stability and hybridization properties of the
RNA duplex. In contrast, attachment of labels on the sugar
edge at positions other than the 20-position of the ribose has
not yet been explored.
The alkyne moiety was introduced via nucleophilic substitu-
tion of the incorporated fluoride by propargylamine. The
subsequent transformation of this propargyl containing
guanosine derivative into its phosphoramidite (Fig. 1a) was
followed by its incorporation onto an siRNA antisense strand
(50-end) during solid phase oligonucleotide synthesis. The click
product of the resulting strand with an Atto 590 azide dye was
HPLC purified and hybridized to a commercially available
sense strand (either unlabeled or containing an Atto 488 dye
on its 30-end), as is illustrated in Fig. 1b.
With the exception of the rare methyl or methylthio-
modifications of adenine on position 2,9–11 guanine is the only
nucleobase that carries naturally occurring modifications on
the sugar edge. Since such modifications occur naturally in
Watson–Crick helices12 this suggests guanosine as a promising
candidate, where chemical labeling at this position may
be expected to minimally alter its hybridization properties.
Reports have been published with regard to the effect of
For validation of our labeling method, structural and func-
tional data were acquired, starting with absorbance melting
measurements of the RNA duplexes. These provided informa-
tion on the effect of the labeling method on the thermal
stability of the duplex. Fig. 2 shows that the temperature-
dependent differential UV-absorption measurements of the
siRNA double strands showed only a very slight difference
in the melting temperatures of labeled strands, as compared to
the unlabeled strands. A small destabilization, caused by the
fluorescent labeling on the 50-end of the RNA, resulted in a
Institute of Pharmacy and Biochemistry, Johannes Gutenberg
University, 5 Staudinger Weg, Mainz, D-55128, Germany.
E-mail: dominol@uni-mainz.de; Fax: +49 6131 39 20373;
Tel: +49 6131 39 24352
w In memory of Professor H. Goni Khorana.
z This article forms part of the ‘Nucleic acids: new life, new materials’
web themed issue.
y Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc34015a
c
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Fig. 1 (a) Eight step synthetic route to the final alkyne functionalized
phosphoramidite (CTDNPI): (i) peracetylation of guanosine, (ii) ether
synthesis via the Mitsunobu reaction, (iii) diazotization of the aromatic
amine and nucleophilic substitution by the fluoride ion,14,19,20
(iv) nucleophilic substitution of the fluoride ion by propargylamine
and concomitant hydrolysis, (v) 50-O-DMT protection, (vi) 20-O-TOM
protection and (vii) 30-O-phosphitylation. (b) Click reaction between
the Atto 590 azide and the alkyne functionalized oligonucleotide,
followed by a hybridization step with the labeled sense strand, showing
reserved base pairing between the modified G and the C of the sense
strand.
Fig. 2 Comparison of the UV melting curves of the unlabeled siRNA
duplex (black) with those of the (a) single labeled and (b) double
labeled constructs.
50-labeling of the antisense strand is controversial,30,31 with
assessments ranging from ‘‘no effect’’31 to ‘‘complete loss of
RNAi activity’’.32–34 Because this type of labeling is impor-
tant for a FRET-based imaging technique of intact siRNA
developed in our lab,29 we decided to quantitatively address
this question by determining IC50-values in an established
reporter gene assay, using the eGFP reporter gene.15,16,29
The expression of the enhanced green fluorescent protein
(eGFP) in HEK cells was monitored by FACS at varying
concentrations of the different siRNA double strands. As
shown in Fig. 3a, attachment of dye to the 30 end of the sense
strand did not alter the IC50. However, attachment to the
50 end of the antisense strand of various dyes, such as TAMRA,
Atto 590 and Atto 647N, via the phosphate (chemical struc-
ture shown in Fig. S7, ESIy) did increase the IC50 by B5-, 10-
and 12-fold, respectively, thereby roughly correlating with the
molecular weight of the dye. Especially the only 5-fold
decrease due to the TAMRA-label shows that labeling at the
50 end is mildly detrimental, but does by no means completely
abolish RNAi activity. As mentioned above, this labeling site
was considered critical because methylation of the 5’OH
ablated RNAi activity.17,18 The question then arises, if the
observed mild impediment results from the labeling chemistry,
i.e. if the attachment of the spacer onto the 50-phosphate
makes a major contribution to the increased IC50. To answer
this question, we used our new phosphoramidite. As shown in
Fig. 3b, the corresponding siRNA, sugar edge-labeled with
Atto 590 surprisingly displays an IC50, which is increased
by a factor of 50 compared to the reference and by a factor
melting temperature difference of a mere 1 1C. In order to
clarify whether this was due to the dye in general, or the
labeling on the sugar edge in particular, a comparison was
made between our construct and an antisense strand labeled
via its 50 end. Resulting similar Tm values of the two constructs
indicate that labeling on the N2 of guanosine has the same
little structural impact as has the labeling on the 50-position,
although the labels are attached at two completely different
sites of the nucleotide. It can be concluded that the observed
effects are due to the presence of the label on the 50-nucleotide
as such and are not associated specifically with our new
labeling position.
In addition, we added an Atto 488 label on the 30-end of the
opposing strand, in order to determine if fluorescent labeling
at this particular domain of siRNA is affecting melting in
general (Fig. 2b). Once again, the Tm did not change upon
introduction of the label. As a result, labeled constructs thus
obtained are suitable for in vivo tracking of intact siRNA
during live cell imaging.29 Imaging of siRNA labeled on the
sugar edge (Fig. S6, ESIy) shows that transfection, cellular
uptake and intracellular distribution are unimpeded.
We applied our new labeling scheme to investigate the effect
of labeling on RNAi activity. Literature on the effect of
c
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c
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Chem. Commun.