Enzyme-free interrogation of RNA sites via primers and oligonucleotides 3′-linked to gold surfaces

Article abstract of DOI:10.1021/ol070724g

The synthesis of a phosphoramidite is described that was used for the preparation of oligonucleotides with a 3′-terminal thiol, linked to the DNA via a SAM-forming undecyl chain and a nonadsorptive tetraethylene glycol unit. A gold surface featuring oligo

Full text of DOI:10.1021/ol070724g

ORGANIC
LETTERS
2007
Vol. 9, No. 11
2187-2190
Enzyme-Free Interrogation of RNA Sites
via Primers and Oligonucleotides
3′-Linked to Gold Surfaces
Ulrich Plutowski,† Stephanie R. Vogel,† Matthias Bauer,‡ Christopher Deck,†
Michael J. Pankratz,‡ and Clemens Richert*,†
Institute of Organic Chemistry, UniVersity of Karlsruhe (TH), 76131 Karlsruhe,
Germany, and Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe,
Postfach 3640, 76021 Karlsruhe, Germany
cr@rrg.uka.de
Received March 24, 2007
ABSTRACT
The synthesis of a phosphoramidite is described that was used for the preparation of oligonucleotides with a 3
′
-terminal thiol, linked to the
DNA via a SAM-forming undecyl chain and a nonadsorptive tetraethylene glycol unit. A gold surface featuring oligonucleotide probes allowed
for label-free in situ mass spectrometric determination of a nucleotide in subpicomole quantities of an RNA transcript.
Of the two polynucleotides, DNA and RNA, the latter is
less well developed in terms of sequencing technology,
amplification, and labeling. The range of biological roles is
more diverse for RNA than DNA, though. Not only is RNA
known to be important as genetic material, e.g., for retro-
viruses, it is also a key component of cellular machineries,
a regulator of gene expression, and the material for ribo-
zymes.1 New functional RNA is being developed through
in vitro selection (SELEX),2,3 and modified RNA is emerging
in therapeutics,4 making novel techniques for the interroga-
tion of RNA desirable.
RNA. Traditionally, enzyme-free extension of RNA has been
slow and has led to product mixtures,5 making it unsuitable
for sequence analysis. New leaving groups for the activated
monomers and “helper” oligonucleotides accelerate these
reactions. Downstream-binding helpers also increase the yield
and ensure formation of a single extension product.6 But,
the quantities required for such assays were too large for
genotyping applications.
(3) Gold, L.; Polisky, B.; Uhlenbeck, O.; Yarus, M. Annu. ReV. Biochem.
1995, 64, 763-797.
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Sebesta, D. P.; Tarasow, T. M.; Willis, M. C.; Zichi, D. A. Bioorg. Med.
Chem. 1997, 5, 1087-1096. (b) Soutschek, J.; Akinc, A.; Bramlage, B.;
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T.; Rajeev, K. G.; Rohl, I.; Toudjarska, I.; Wang, G.; Wuschko, S.; Bumcrot,
D.; Koteliansky, V.; Limmer, S.; Manoharan, M.; Vornlocher, H. P. Nature
2004, 432, 173-178.
We became interested in enzyme-free primer extension
as an inexpensive method for determining nucleotides in
† University of Karlsruhe.
‡ Forschungszentrum Karlsruhe.
(1) (a) Cech, T. R. Angew. Chem., Int. Ed. Engl. 1990, 29, 759-768.
(b) Altman, S. Angew. Chem., Int. Ed. Engl. 1990, 29, 749-758.
(2) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822.
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10.1021/ol070724g CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/05/2007

Mass spectrometry as read-out does not require labeling
and produces more specific signals than fluorescence. In situ
mass spectrometric detection of short DNA strands hybrid-
ized to probe sequences on gold surfaces has recently been
shown,7 including nonenzymatically extended primers.8 This
work was limited to four-strand systems, short DNA
templates, and required 3′-amino-2′,3′-dideoxynucleosides at
the 3′-terminus of the primer (Figure 1a). We wished to make
nanoparticles13 that aggregate upon hybridization of DNA
sequences.14 Complexes between DNA and cationically
modified gold clusters release the DNA in glutathione-
dependent fashion,15 and a release can also be induced
photochemically.16
Unlike common bifunctional linkers for the immobilization
of oligonucleotides that are attached to the 5′-terminus, our
system required a 3′-appended linker. This linker cannot be
readily introduced at the end of conventional DNA syntheses
where the 3′-terminus is bonded to the solid support. To the
best of our knowledge, no method existed for preparing DNA
with a 3′-appended, thiol-terminated linker consisting of a
long alkyl chain for formation of a self-assembled monolayer
(SAM) and a distal oligoethyleneglycol portion reducing
nonspecific adsorption. But, methods for other 3′-thiol-
bearing DNAs have appeared in the literature, including one
for the synthesis of oligonucleotides with a 3′-terminal
thymidine residue featuring an alkylthiol moiety,17 one for
3′-disulfide functionalized DNA with an amide linkage,18 and
one involving controlled pore glass (cpg) with a disulfide
and a butyl spacer.19 Disulfide-derivatized cpg with propyl
or hexyl chains is used for the preparation of DNA attached
to gold nanoparticles.20
The route to our reagent for the preparation of DNA with
a 3′-appended thiol linker is shown in Scheme 1. The linker
Scheme 1. Synthesis of Phosphoramidite Linker Reagent
Figure 1. Components of chemical primer extension assays for
determining nucleotides in template strands: (a) known system with
a short DNA template and 4 strands and (b) three-strand system
with a long RNA transcript as the template.
base calls in subpicomole quantities of RNA. RNA is less
prone to fragment under MALDI conditions than DNA.9 We
designed a three-strand system with an RNA transcript as
template, a primer, and a 3′-linked oligonucleotide that serves
as a capture strand and as a helper (Figure 1b).
Planar surfaces allow for interrogating templates in arrayed
format. Gold readily reacts with thiols or disulfides in organic
or aqueous solutions, but few other functional groups.10 Gold
is also suitable as a target surface for MALDI-TOF MS,
allowing for sensitive detection of noncovalently bound
molecules.11,7 Further, gold allows for surface plasmon
resonance monitoring,12 and may be obtained in the form of
is structurally related to those used by Whitesides,21 Mrk-
sich,22 and others.7 Starting material 1 was prepared from
1-bromoundec-10-ene in three steps.21 Mixed disulfide 1 was
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625.
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Kristiansen, K.; Roepstorff, P. Nucleic Acid Res. 1993, 21, 3347-3357.
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17-48.
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Nygren, P.-A. Anal. Biochem. 1997, 246, 34-44.
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J. Chem. Soc., Chem. Commun. 1994, 801-802.
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Org. Lett., Vol. 9, No. 11, 2007

The synthesis of linker-bearing oligonucleotides (Scheme
2) started from cpg loaded with a nucleoside (6), i.e., an
inexpensive commercial support for DNA synthesis. A
coupling cycle with phosphoramidite 5 yielded 7, which was
subjected to conventional DNA chain assembly to give
protected oligonucleotide 8. Extensive capping of residual
unreacted hydroxy groups after the initial coupling step
involving 5 prevented the formation of linker-free DNA
chains that complicate purification of the final product.
Treatment of 8 with aqueous ammonia containing 1,4-D,L-
dithiothreitol removed the nucleobase and backbone protec-
tion groups, and cleaved the disulfide bridge to give 9, whose
5′-dimethoxytrityl group facilitated purification. A cartridge
step with on-support detritylation gave target oligonucleotide
10 in high purity.
Scheme 2. Synthesis of DNA with the 3′-Attached Linker
Gold-coated substrates were smoothed in an H2 flame, and
solutions of 10 were applied to individual spots, followed
by coating the remaining free surface by treatment with
disulfide reagent 117 (Scheme 3). A mixture of RNA primer
5′-CCACAACCCA-3′ (12, 800 fmol) and 376 nucleotide-
long RNA 13 (100-500 fmol) was then hybridized to the
DNA-displaying spots. Template 13 is a partial transcript
of the gene encoding transcription factor cdx4 in zebrafish
(Danio rerio). After a brief wash with NH4OAc buffer, a
solution of the oxyazabenzotriazolides of the four ribonu-
cleoside-5′-monophosphates (A, C, G, and T; 14a-t, 6 mM
each)23 in reaction buffer was added to each spot. Primer
extension was allowed to occur at room temperature in a
humid chamber with application of fresh monomers after
10 and 23 h. After 27 h, MALDI matrix was applied, and
the extended primer was detected spectrometrically (Figure
2). Near-quantitative primer extension with good base selec-
tivity for G as templating base was seen down to 100 fmol
of the template (Table 1). Other bases are known to template
selectively.6 Reactions with the same experimental setup, but
O-protected with the dimethoxytrityl group (DMT) com-
monly used during DNA chain assembly to give 2 in 78%
yield (see the Supporting Information for protocols). The
subsequent thiopyridone-releasing disulfide exchange with
thiol 3 gave 4 in 88% yield in slow reactions, even under
basic conditions. Phosphitylation of 4 gave phosphoramidite
5 in 77% yield. This, like its precursors, was purified by
chromatography on silica. Attempts to isolate it by precipita-
tion, a common purification strategy for nucleosidic phos-
phoramidites, remained unsuccessful.
Scheme 3. Generating Arrays of Capture Oligonucleotides on SAM-Coated Gold Surfaces and Primer Extension Templated by RNA
Org. Lett., Vol. 9, No. 11, 2007
2189

Table 1. Results from Primer Extension with 12 and 16
quantity of
sequence selectivitya
primer
RNA (12)
RNA (12)
amino-DNA (16)
template (fmol)
+C/+T/+A/+G
100
500
500
>3:1:1:1b
76:13:7:4b
>99:1:1:1c
a Product distribution for extended primer. b After 27 h. c After 4 h.
suitable for direct genotyping of individual nucleotides mass
spectrometrically in RNA transcripts. An RNA transcript
with several hundred nucleotides was shown not to interfere
with MALDI-TOF MS. Our method requires only 3 strands
and has high sensitivity, which may be improved further by
miniaturization. The methodology may become useful for
genotyping tumors or viruses24 mass spectrometrically,25
particularly if RNA used for quantitative expression analysis
or identification of viruses via microarrays26 is simulta-
neously genotyped, providing key information on geno- and
haplotypes. Approximately 80% of all known virus are
retroviruses, such as HIV, Ebola virus, influenza virus,
including the H5N1 strain, arbovirus-B, and the hepatitis
viruses. Cancer diagnosis will benefit from expression
analysis of oncogenes efficiently combined with SNPs
detection.27
Figure 2. MALDI mass spectrum of surface-bound RNA primer
12 extended to 15, in an enzyme-free reaction templated by RNA
transcript 13 on a gold slide with 10; 500 fmol of template and
800 fmol primer were hybridized to the surface.
DNA-primer 16 with a 3′-terminal 3′-amino-2′,3′-dideoxy-
adenosine residue and the activated ribonucleotides occurred
faster, with full primer conversion in good base fidelity in
<4 h.
In conclusion, we report gold surfaces coated with self-
assembled monolayers displaying 3′-linked oligonucleotides
Acknowledgment. The authors thank Dr. E. Kervio (U.
Karlsruhe) for helpful discussions. This work was supported
by DFG (grant No. 1063/1-4 and FOR 434).
Supporting Information Available: Syntheses, template
sequence, protocols for gold substrate and its use, and NMR
and MS spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
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OL070724G
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