A 5′-cap for DNA probes binding RNA target strands

Article abstract of DOI:10.1002/chem.201101828

Detecting short RNA strands with high fidelity at any of the bases of their sequence, including the termini, can be challenging, since fraying, wobbling, and refolding all compete with canonical base pairing. We performed a search for 5′-substituents of o

Full text of DOI:10.1002/chem.201101828

FULL PAPER
DOI: 10.1002/chem.201101828
A 5’-Cap for DNA Probes Binding RNA Target Strands
Simone Egetenmeyer and Clemens Richert*[a]
Abstract: Detecting short RNA strands
cap that stabilizes any of the four can-
onical base pairs, with DTm values of
up to +13.18C for an octamer. At the
same time, the cap increases discrimi-
nation against any of the 12 possible
terminal mismatches, including mis-
matches that are more stable than their
perfectly matched counterparts in the
control duplex, such as A:A. A probe
with the cap also showed increased se-
with high fidelity at any of the bases of
their sequence, including the termini,
can be challenging, since fraying, wob-
bling, and refolding all compete with
canonical base pairing. We performed
a search for 5’-substituents of oligo-
deoxynucleotides that increase base
pairing fidelity at the terminus of du-
plexes with RNA target strands. From
a total of over 70 caps, differing in
stacking moiety and linker, a phospho-
diester-linked sequence of the residues
of l-prolinol, glycine, and oxolinic acid,
dubbed ogOA, was identified as a 5’-
lectivity in the detection of two closely
related microRNAs, let7c and let7a,
with a DTm value of 9.28C. Melting
curves also yielded thermodynamic
data that shed light on the uniformity
of molecular recognition in the se-
quence space of DNA:DNA and
DNA:RNA duplexes. Hybridization
probes with fidelity-enhancing caps
should find applications in the individ-
ual and parallel detection of biological-
ly active RNA species.
Keywords: DNA
·
hybridization
probe · molecular caps · molecular
recognition · RNA · RNA recogni-
tion
Introduction
zation is also required for many modern methods of mas-
sively parallel or ultrasensitive detection.[8] Molecular recog-
nition through hybridization is challenging when the target
has a high propensity to fold and to form alternative base
pairs, such as G:U or U:G wobble base pairs, and when the
sequence differences are in terminal regions, where fraying
and wobbling is common.
Of the two canonical nucleic acids, RNA is more abundant
in the cell, shows greater diversity in its functions, and has a
greater propensity to fold into complex three-dimensional
structures.[1] Whereas the sole role of DNA is to store genet-
ic information, ribonucleotides have a host of roles, includ-
ing forming the core components of the translational ma-
Hybridization probes for the detection of RNAs are most
frequently oligodeoxynucleotides, as DNA is less expensive
to synthesize than RNA. Oligodeoxynucleotides are also
chemically and enzymatically more stable than oligoribonu-
cleotides. So, the most relevant type of probes for the detec-
tion of microRNAs are DNA strands that form DNA:RNA
hybrid duplexes upon recognizing their target. If the level of
affinity and selectivity required is not met by unmodified
DNA, modified oligodeoxynucleotide probes are called for.
Increasing target affinity and selectivity beyond that of natu-
ral DNA may be achieved with different approaches. The
modifications may involve the backbone of the probe, as in
locked nucleic acids (LNAs),[9] peptide nucleic acids
(PNAs),[10] phosphoramidates,[11] or oligonucleotides con-
taining 2’-fluoronucleosides.[12] Alternatively, the nucleo-
ACHTUNGTRENNUNGchinACHTUNGTRENNUNGery, acting as the recognition motif in cofactors, and reg-
ulating gene expression through riboswitches[2] or micro-
RNAs.[3–6] Understanding the biological roles of RNAs re-
quires analytical methods for detecting them selectively,
often against a genomic background. Highly selective detec-
tion is particularly important for short RNAs with roles in
the regulation of gene expression, such as microRNAs, for
which accuracy in the detection can be critical for medical
diagnostics. Being short, many microRNAs do not lend
themselves to routine amplification through reverse tran-
scription and the polymerase chain reaction (PCR),[7] so that
high fidelity hybridization is critical. Due to the abundance
of closely related isoforms, high sequence selectivity is re-
quired at any given position of the duplex. Accurate hybridi-
AHCTUNGTRENNUNG
bases may be modified[13,14] or substituents that aid the inter-
rogation of the target structure may be introduced in the in-
terior of the probes. Substituents may also be introduced at
the termini of oligonucleotides.[15,16] ꢀHighly decoratedꢁ hy-
bridization probes use a combination of 5’- and 3’-caps, as
well as substituents in the interior of the sequence.[17] Such
probes were introduced to aid the massively parallel detec-
tion of diverse DNA target sequences on microarrays by
smoothing out the differences in duplex stability between
[a] Dipl. Chem. S. Egetenmeyer, Prof. C. Richert
Institut fꢂr Organische Chemie
Universitꢃt Stuttgart, 70569 Stuttgart (Germany)
Fax : (+49)711-685-64321
E-mail: lehrstuhl-2@oc.uni-stuttgart.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101828.
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duplexes with different G/C content[18] and ensuring high
base pairing fidelity up to the very termini. It is desirable to
also realize the potential of highly decorated probes for
RNA detection.
Only a modest number of covalently attached ligands or
ꢀcapsꢁ are known that increase target selectivity at the termi-
nus of duplexes between probes and target strands.[19] We re-
cently reported a cap for the 3’-terminus of DNA hybridiza-
tion probes for binding RNA target strands,[20] as well as a
number of caps for the 5’-terminus of probes binding DNA
target strands.[16] However, no cap for the 5’-terminus of hy-
bridization probes that increases fidelity in the recognition
of RNA targets had been identified. Caps commercialized
for the 5’-terminus of DNA probes binding DNA tar-
ACHTUNGTRENNUNG
gets[19e,21] did not perform well when binding RNA target
strands (see below). As a consequence, we initiated a search
for 5’-caps on oligodeoxynucleotides that bind RNA targets.
Here, we present the results of this search, together with
data on the thermodynamics of duplex formation and base
pairing fidelity at the terminus.
Figure 1. Difference between melting points of DNA:DNA duplexes and
DNA:RNA duplexes with terminal mismatch sequences and their corre-
sponding perfectly matched (PM) duplexes. The DNA:DNA duplexes
Results
Effect of the backbone on pairing selectivity at the termi-
nus: Earlier work on molecular caps for the termini of hy-
bridization probes has shown that the duplex-stabilizing
effect depends on the backbone structure of the target
strand.[19b] At first glance, this may seem surprising, as the
base pairs formed are identical (G:C) or near-identical (A:T
versus A:U) for DNA:DNA and DNA:RNA duplexes. If
the caps were interacting predominantly with the nucleo-
are d
nucleotides given on the bars. The DNA:RNA duplexes are
(XGGTTGAC):r(GUCAACCY). Conditions: phosphate buffer (10 mm,
ACHUTGTNRNEUG(N XGGTTGAC):dCAHTUNGTRNE(NUGN GTCAACCY) in which X and Y stand for the
d
G
ACHTUNGTRENNUNG
pH 7), NaCl (1m), and 1.5 mm strand concentration, ldet =260 nm.
with terminal T, did the DNA:RNA cases show more selec-
tivity in hybridization than their DNA:DNA counterparts.
To better understand the molecular basis of base pairing
fidelity and the effect of the backbone structure (ribonucleo-
sides versus deoxyribonucleosides), we decided to extract
thermodynamic data from melting curves of duplexes with
or without single mismatches at the termini. For this, we
used both the current data (Figure 1) and the original melt-
ing curves from our earlier work on 3’-caps.[23,20] Other melt-
ing point data for longer duplexes with single mismatches
(both DNA and RNA), determined on microarrays, can be
found in the literature.[24] The enthalpy and entropy of
duplex formation were extracted by using the program Melt-
win.[25] For each duplex, four individual curves were ana-
lyzed and the standard deviation was calculated. The data
for each of the four cases (5’-terminus and 3’-terminus for
both DNA and RNA target strands) are shown in graphical
form in Figure 2a–d. The numerical values can also be
found in Tables S6–S9 in the Supporting Information.
As is common for nucleic acid duplexes, there is a very
significant enthalpy–entropy compensation. The change of a
single base in an octamer duplex does not lead to dramatic
changes in the thermodynamic characteristics of the duplex
to single-strand transition. Nevertheless, there are some
noteworthy trends that can be gleaned from the DH and
TDS values that together make up the DG of duplex forma-
tion for each of the duplexes. Firstly, in the vast majority of
ACHTUNGTRENNUNG
bases of the terminal base pair,[22] little selectivity should
result. So, either the caps were strongly interacting with the
backbone or were further enhancing an intrinsically lower
stability and selectivity of base pairing at the termini of
DNA:RNA duplexes.
To shed light on this issue for our present case of
DNA:RNA duplexes, we first measured the melting points
of octamer duplexes with all of the 16 possible combinations
of nucleobases at the 5’-terminus. For this, we used the
DNA probe d
and DNA or RNA target strands d
(GUCAACCY), in which Y at the 3’-terminus is A, C, G,
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
rACHTUNGTRENNUNG
T, or U. Figure 1 shows the drops in melting point induced
by mismatches at the terminus for either type of duplex.
The individual absolute Tm values can be found in Tables S1
and S2 in the Supporting Information. For seven out of
twelve cases, the mismatch discrimination was found to be
poorer for RNA compared with DNA as the target. The dif-
ference was particularly pronounced for the problematic
cases, such as those with A at the 5’-terminus of the probe,
for which non-Watson–Crick combinations A:A and A:G
result in more stable mismatched DNA:RNA duplexes than
the fully complementary duplexes with canonical base pair-
ing throughout. Only for the most selective pairings, involv-
ing C as the 5’-terminal base of the probe, and one pairing
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DNA Probes for Binding RNA Targets
FULL PAPER
Figure 2. Thermodynamic parameters of duplex formation at 378C, as determined from melting curve data by using Meltwin v3.5:[26] a) for the 16
DNA:DNA duplexes d(AGGTTGAX) and d(YTCAACCT), in which X and Y stand for the nucleotides given above and below the respective bars;
b) for the 16 DNA:RNA duplexes of (AGGTTGAX) and (YUCAACCU); c) for the 16 DNA:DNA duplexes of (XGGTTGAC) and
(GTCAACCY); and d) for the 16 DNA:RNA duplexes of d(XGGTTGAC) and r(GUCAACCY). The columns in the upper part of each graph show
A
ACHTUNGTRENNUNG
d
A
r
A
dACHTUNGTRENNUNG
d
N
N
ACHTUNGTRENNUNG
DH values and those in the lower part show TDS values. Thermodynamic data were determined from melting curves detected at 260 nm from a sodium
phosphate buffer (10 mm) with NaCl (1m) and 1.5 mm strand concentration. Note the y-axis expansion, chosen to highlight differences.
all cases, the duplexes with perfectly matched bases experi-
ence the greatest enthalpic gain upon duplex formation, ac-
companied by the most significant entropic loss, compared
with the mismatch-containing combinations. Secondly, in all
but three cases, the enthalpic gain upon duplex formation is
smaller when RNA is the target strand than when DNA is
the target strand of the octamer probe. Thirdly, the A:A or
G:A purine:purine combinations and the G:U wobble base
pair are particularly stable cases for the DNA:RNA duplex
when a mismatch is found at the 5’-terminus of the probe
and the 3’-terminus of the target. For this, the lowest fidelity
situation, purines at the 5’-terminus of the probe give the
most similar DH and TDS values for perfectly matched and
mismatch-containing duplexes, that is, the lowest intrinsic
capability to generate free energy differences as the basis of
selectivity in molecular recognition. Other than this, there
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S. Egetenmeyer and C. Richert
are few truly exceptional compensation phenomena, though,
that show an unusually strong enthalpic gain for a mis-
matched base pairing or a very strong entropic loss upon
formation of a canonical base pair. Rather, the formation of
a base pair appears to provide an enthalpic gain, whereas
the presumed lack of pairing makes duplex formation both
enthalpically less favorable and entropically less costly.
However, the data in Figure 1 also show that the base
pairing fidelity at the terminus depends strongly on the nu-
cleobase to be detected. For C at the 5’-terminus of the
probe, drops in the melting point, caused by a mismatched
base in the target, are greatest, whereas for A at the 5’-ter-
minus, discrimination is poorest. When selectivity is poor in
the DNA:DNA case, it is poorer still in the DNA:RNA case
with RNA as the target strand. As mentioned above, both
of the mismatched purine bases in the target give a higher
melting point for the duplex with A at the 5’-terminus of the
probe than with the matched base, U. This confirms just
how difficult it is to achieve fidelity at the 3’-terminus of an
RNA target. Whilein the majority of the mismatched cases
the DNA:DNA duplex gives a greater enthalpy of forma-
tion, the A:A pairing gives a larger DH for the DNA:RNA
case. This suggests that there are stabilizing interactions not
found in sequences with other terminal bases. Inspection of
Figures 2c and 2d, in which the corresponding data are pre-
sented for binding the other terminus (the 5’-terminus) of
the DNA:RNA duplex, makes the phenomenon more con-
spicuous still. Here, the duplexes with an RNA target strand
give lower enthalpy values for duplex formation throughout,
even when A is the terminal nucleobase.
It is unlikely that hydrogen bonding is at the core of this
difference in selectivity for the two backbones. Rather, dif-
ferences in the stacking interactions are probably causing
the effect. We suspected that the exceptionally high stability
of duplexes with 5’-terminal A facing a mismatched A or G
in the RNA target was due to bridging interactions that the
purine base provides when stacking on the neighboring base
pair of the duplex. When RNA is the target strand, an A-
form duplex can be expected to form, whereas DNA:DNA
duplexes prefer the B-form.[1] The A-form helix possesses a
smaller helical pitch (rise per turn) and a smaller helical
twist per base pair. This affects the extent to which neigh-
boring bases stack on the nucleobases of a given base pair.
We modeled this situation for terminal nucleotides, as-
suming that the dangling residue adopts the standard confor-
mation of A- or B-type helices. Figure 3 shows the extent to
which a 5’-terminal adenine bridges the duplex in A- and B-
form geometries, respectively. A similar image that shows a
larger portion of the respective duplexes can be found in the
Supporting Information (Figure S1). The adenine bridges
the penultimate base pair more extensively in the A-type
duplex, making the presence of the complementary base of
the target strand less significant for duplex stability. A more
detailed analysis of the effect of dangling residues on the
stability of DNA and RNA duplexes can be found in the
recent literature.[27] This analysis, building on a significant
body of earlier work,[28,29] suggests that a dangling 3’-termi-
Figure 3. Modeling of the bridging effect of the nucleobase of dangling A
residues at the 5’-terminus of duplexes in standard B- or A-form, as ex-
pected for DNA:DNA and DNA:RNA duplexes, respectively. The stack-
ing arrangement of the nucleobase of the dangling A (dark grey) and the
terminal base pair (light grey) are shown, from a view point along the
helix axis. a) Terminus of a DNA:DNA duplex, as generated in silico for
the duplex of 5’-ACGA-3’ and 5’-TCG-3’. Coordinates were generated
with the structure-building tool in Macromodel (version maestro 7.5.106),
using default parameters for A-form duplexes. b) Terminus of
a
DNA:DNA duplex, as generated in silico for the duplex of 5’-ACGA-3’
and 5’-UCG-3’ in B-form. Note that for the B-form, the dangling A resi-
due bridges the duplex less than for the A-form.
nal residue of an RNA duplex provides the strongest
duplex-stabilizing effect, compared with other dangling nu-
cleotides, so that a stacked 3’-terminal A residue on the
target may, by itself, provide much of what a Watson–Crick
base pair would provide in duplex-stabilizing effect.
The shielding effect of 3’-dangling purines on RNA has
also been found by NMR[30] and time-resolved spectrosco-
py.[31] The more strongly bridging the stacking interactions
of the 5’-terminal purine base,[28d] and the smaller the re-
maining stacking surface for the pyrimidine base of the
target strand, the less likely it is that formation of the cor-
rect, canonical base pair will significantly increase duplex
stability. If the 3’-terminal A of the target strand further
stacks onto the 5’-terminal A of the probe strand, a zipper-
like stacking arrangement could result that reduces the like-
lihood of duplex dissociation, further adding to what 3’-dan-
gling residues contribute to stability.[32]
Independent of the cause of the poor mismatch discrimi-
nation, the lack of selectivity in base pairing for duplexes
with a purine at the 5’-terminus of the probe motivates a
search for caps as fidelity-enhancing elements more than
other constellations in the sequence space of nucleic acids.
The cap to be identified should be large enough to provide
additional enthalpic gains upon duplex formation. Ideally,
the cap should also be universal, stabilizing any of the four
canonical base pairs, not just duplexes in which the 5’-termi-
nal base of the probe is a purine, that is, the most difficult
case, even for DNA:DNA duplexes.[21] Since destabilizing
mismatched pairs is unrealistic, increases in selectivity can
most likely be achieved with caps that bind tightly on canon-
ical base pairs, but poorly on mismatched base pairs with
different geometries, such as G:U wobble base pairs.[33]
One way to design a cap is to use a dangling C-nucleoside
with a large aromatic ring as a base surrogate. Such C-nu-
cleosides are known for a number of annelated aromatic
ring systems, most notably pyrene, and we decided to use a
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DNA Probes for Binding RNA Targets
FULL PAPER
pyrenyl-C-nucleoside[15d,34] as one benchmark compound for
our 5’-caps. The other benchmark was an unmodified dan-
gling deoxyadenosine residue.
Unless a cap showed a significant effect on the duplexes of
1 with the complementary RNA strand, it would not be se-
lected for the second phase, during which optimization was
to be performed on an octamer sequence. The shorter
duplex resolves more subtle changes in stability, whereas the
longer strand gives duplexes more representative of those
typically formed on microarrays. All caps feature a terminal
Synthesis and selection of probes with 5’-caps: Scheme 1,
parts a and b show the general design of our cap-bearing
strands. Scheme 1c shows the synthesis of the first series of
DNA strands with 5’-caps. We selected all-A/T tetradecamer
1 for this series, as a particularly challenging case for the
recognition of target strands, forming weak base pairs
throughout. The same sequence has previously been em-
ployed in our study on isostable DNA:DNA duplexes.[17]
stacking moiety for interacting with the nucleoACTHNUTRGNEUGbN ases and a
linker unit for connecting the stacking moiety and the phos-
phodiester at the 5’-terminus of the probe strand. We opted
for a flexible alkyl chain to screen for the proper length of
the linker. Besides the stacking moiety and linker, the back-
Scheme 1. Cap-bearing oligonucleotides: a) cartoon representation of a duplex with cap, b) retrosynthetic considerations for the assembly of oligonucleo-
tides with caps, and c) synthesis of 5’-capped DNA strands with an all-A/T sequence. Bz=benzoyl; MMT=monomethoxytrityl; TCA=trichloroacetic
acid; HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; DIEA=N,N-diisopropylethylamine.
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S. Egetenmeyer and C. Richert
bone of the 5’-terminal nucleotide of the probe was to be
varied, with some members of our small library featuring a
5’-terminal 2’-deoxy-2’-fluorouridine residue (general struc-
ture 2), that is, a substitute for thymidine considered to be
particularly well suited for binding RNA targets.[12,35]
The assembly of the cap-bearing oligonucleotides started
with a conventional DNA synthesis on controlled pore glass,
producing supports 3 and 4. Then, one of five different
linker phosphoramidites 5a–e, with alkyl chains 2–6 methyl-
ene groups in length,[36] was coupled to the support (most
frequently 5b), followed by oxidation, yielding supports 6a–
e or 7b–d. The MMT groups were removed, and the carbox-
ylic acids of potential stacking moieties were coupled under
peptide coupling conditions. Cleavage from the solid sup-
port with concomitant removal of all protecting groups by
treatment with saturated aqueous ammonia gave crude
products that were purified by RP–HPLC.
The use of the amino terminal linker allowed for testing
of a range of residues of different carboxylic acids as stack-
ing moieties with modest synthetic effort. The stacking moi-
eties chosen are shown in Scheme 2. Some are known to act
as molecular caps for DNA:DNA duplexes,[16] others were
selected from commercially available carboxylic acids ac-
cording to the following criteria: 1) the resulting stacking
moiety should contain two or more rings for strong stacking
interactions with the terminal nucleobases of the
DNA:RNA duplex; 2) the acyl residue should contain
favorable dipole–dipole interactions with the duplex termi-
nus; and 3) the functional groups should be compatible with
oligonucleotide synthesis. The short-hand names of the re-
sulting cap-bearing strands start with the number of the
DNA sequence, followed by a lower-case letter (or letters)
for the linker and two upper-case letters for the stacking
moiety. For example, 1bMS stands for sequence 1 featuring
propyl linker “b”, to which the acyl group of trimethoxystil-
bene, “MS”, is appended (a=ethyl, b=propyl, c=butyl, d=
pentyl, e=hexyl, f=aminoethoxyethyl, t=5’-amino-5’-de-
AHCTUNGTRENNUNG
ture-known or commercial molecular caps were coupled di-
rectly to 3, without the use of a separate linker (Scheme 3).
This included the phosphoramidite of pyrenylmethylpyrroli-
dine (8),[21] pyrenylbutanol (9),[37] an acridine (10), anthra-
quinoylamido uridine (11),[20] and pyrenyl-C-nucleoside
(12),[34] our benchmark compound (see Figure S2 in the Sup-
porting Information for the full structures of 11 and 12).
Coupling and oxidation were again followed by deprotection
in aqueous ammonia. In the case of the pyrenyl-C-nucleo-
side, the 5’-hydroxy group was deprotected after HPLC to
release fully deprotected pentadecamer 1PC, whereas for
1AC, the DMT group was left in place to allow for stacking
interactions.
Table 1 lists the UV melting points of duplexes of DNA
tetradecamers of general structure 1 with the fully comple-
mentary RNA target strand. Compared with the unmodified
heteroACHTUNGTRENNUNGatoms to avoid excessive lipophilicity and to allow for
Scheme 2. Carboxylic acid residues of the stacking moieties of molecular caps.
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DNA Probes for Binding RNA Targets
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Table 1. UV melting points for duplexes of DNA tetradecamers of gen-
eral structure
GUC).
1 and RNA target strand r(AUUAUUAAAAAUUA-
DNA strand Melting point [8C][a] DTm [8C][b] Hyperchromicity [%][c]
1[d]
26.5Æ1.1
28.5Æ0.8
29.1Æ0.9
27.5Æ0.9
26.1Æ1.4
22.9Æ0.7
29.6Æ0.8
27.0Æ0.9
27.1Æ0.7
28.5Æ0.7
27.8Æ0.9
29.0Æ0.8
26.8Æ1.3
25.5Æ0.5
27.5Æ0.5
26.4Æ1.0
25.0Æ0.6
26.0Æ1.0
22.2Æ1.0
28.0Æ1.1
27.3Æ0.8
27.0Æ1.0
27.0Æ1.0
26.3Æ0.8
26.0Æ1.0
25.5Æ1.0
26.3Æ1.1
25.7Æ1.0
25.0Æ0.9
25.4Æ0.9
25.1Æ1.0
29.6Æ0.7
28.1Æ0.8
26.9Æ1.0
30.7Æ1.0
27.9Æ1.0
30.8Æ0.8
31.7Æ0.7
36.2Æ0.8
–
2.0
2.6
1.0
À0.4
À3.6
3.1
0.5
0.6
2.0
1.8
2.5
0.3
29
28
27
31
23
14
29
30
28
24
27
30
32
30
33
28
31
33
23
31
34
34
35
32
32
31
32
30
31
26
24
31
30
32
30
30
31
31
28
1A[e]
1AC
1bMS
1PB
1PP
1bCR
1 fCR
1tCR
1bAQ
1tAQ
1UA
1bPQ
1bAT
1bPY
1tPY
1bCH
1bCD
1tBP
1bMN
1bIA
1bPT
1bFL
1bPA
1bNP
1bCT
1bDC
1bPE
1 fPE
1bCO
1 fCO
1bCI
1bNA
1bOF
1bOA
1 fOA
1tOA
1PC
À1.0
1.0
À0.1
À1.5
À0.5
À4.3
1.5
0.8
0.5
0.5
À0.2
À0.5
À1.0
À0.2
À0.8
À1.5
À1.1
À1.4
3.1
1.6
0.4
4.2
1.4
4.3
5.2
9.7
1ogOA
[a] Average of four melting points at 1 mm strand concentration in phos-
phate buffer (10 mm, pH 7) and NaCl (1m). [b] Melting point difference
to unmodified control duplex of
1 with r(AUUAUUAAAAAUUA-
GUC). [c] Hyperchromicity of the thermal transition, measured at
260 nm. [d] Unmodified DNA control strand. [e] Unmodified DNA
strand d(ATAATTTTTAATAAT) with dangling dA residue at the 5’-ter-
minus.
Scheme 3. Synthesis of oligonucleotides with caps introduced through a
single phosphoramidite coupling. DMT=4,4’-dimethoxytrityl.
duplex, most duplexes of cap-bearing strands gave a modest
increase in melting point. However, in a few cases, the DTm
value was negative, suggesting that the cap reduced the pro-
pensity of the DNA strand to engage in duplex formation,
possibly by favoring alternative structures or interfering
with base pairing. The DTm value for the duplex featuring
benchmark pyrenyl-C-nucleoside 1PC was found to be
+5.28C. The otherwise unmodified DNA:RNA duplex with
a 5’-dangling dA residue as the molecular cap (1A) gave an
increase in melting point of +2.08C, that is, less than could
have been expected based on the data on longer RNA du-
plexes with dangling residues.[38] Melting point increases for
1bPY, 1bAQ, and 1bMS were no more than 1.0–2.08C.
Strands featuring caps AC, PP, PB, and UA, introduced in
one step as phosphoramidites, induced no more than a small
melting point increase. This was also true for caps known to
strongly stabilize DNA:DNA duplexes, like pyrenylmethyl-
pyrrolidine (PP),[21] anthraquinone-bearing uridine (UA),[20]
and trimethoxystilbene (MS),[19e] which all had a small effect
on the stability of the DNA:RNA duplex studied. For exam-
ple, the duplex of trimethoxystilbene-bearing 1bMS with
the RNA complement gave a DTm value of +1.08C. The
corresponding DNA:DNA duplex of the same length gives
+5.48C under the same conditions.[17] This demonstrates the
strong effect of the backbone of the target strand on the sta-
bilizing effect of caps. However, the salt dependence of the
melting point did not show unusual effects (see Table S3 in
the Supporting Information for melting points at 150 mm
Chem. Eur. J. 2011, 17, 11813 – 11827
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11819

S. Egetenmeyer and C. Richert
NaCl), and subsequent experiments were run at 1m NaCl
only, as microarray experiments are typically performed at
high salt concentrations.[39]
Chrysene has previously been proposed as a particularly
well-suited stacking moiety.[22b] The duplex of chrysene-bear-
ing 1bCR gave a DTm value of +3.18C, but other large
polycyclic aromatic ring systems, like perylene, coronene,
tert-butylpyrene, anthracene, and 2-phenyl-4-quinoline, did
not stabilize the DNA:RNA duplex (strands 1bPE, 1bCO,
1tBP, 1bAT, and 1bPQ, respectively). Bile acid residues
(1bCH and 1bCD) also failed to increase duplex melting
points. Most stacking moieties with just two aromatic rings
(1bMN, 1bIA, 1bPT, 1bFL, 1bPA, 1bNP, 1bCT, and
1bDC) also fared poorly. The most encouraging results were
obtained for chinolones as stacking moieties. The duplex
with the residue of oxolinic acid (1bOA) gave a DTm value
of +4.28C, just shy of the value for the pyrenyl benchmark
cap. The structural details of the chinolone ring system did
have an effect, though, with cinoxacine inducing a DTm
value of +3.18C (1bCI, isoelectronic to 1bOA), whereas bi-
cyclic nalidixic acid (1bNA) and tetracyclic ofloxacine
(1bOF) provided little duplex stabilization.
Table 2 lists melting points of duplexes of eight other ver-
sions of 1 or 2 with the complementary RNA target strand.
Two features were varied among these probes: the length of
the alkyl chain of the linker (1aOA–1eOA) and the struc-
ture of the 5’-terminal nucleotide residue (2bOA–2dOA).
For conventional, 5’-thymidyl DNA 1, the optimum linker
length, as defined by the highest melting point, was four
(1cOA) or five methylene units (1dOA). For the strands
with a 5’-terminal 2’-fluorouridine (2), higher overall melting
values were recorded, and slightly longer alkyl chains ap-
peared more favorable (Figure 4). The additional increase in
duplex melting point when the 5’-terminal thymidine was re-
Figure 4. Representative melting curves of duplexes with or without a
molecular cap. Perfectly matched DNA:RNA duplexes with RNA target
strand r(CUGAUUAAAAAUUAUUA) and DNA strands d(TAATTTT-
~
&
^
TAATAAT) (1, , control) or cap-bearing 1dOA ( ) and 2dOA ( ).
selves further, as this would require four different cap phos-
phoramidites, one for each possible 5’-terminal base (A/C/
G/T), in probes to be synthesized for practical applications,
making the synthesis more costly and the use of caps less at-
tractive.
Optimization of the linker of the 5’-cap: We then proceeded
to vary the linker structure, using the residue of oxolinic
acid as the stacking moiety. First, two heteroatom-containing
linkages were tested, by using phosphoramidites 13 and 14
(Scheme 4). One features a flexible backbone and a single
oxygen atom in the chain (1 fOA), and one the 5’-amino-5’-
deoxythymidine linker known from the ꢀcomposite capꢁ of
our earlier work on DNA:DNA duplexes (1tOA).[40] The
former gave an inferior duplex-stabilizing effect, compared
to its alkyl counterpart 1bOA (Table 1). The latter gave a
DTm value similar to that of the duplex of 1bOA, even
though the linkers are not structurally similar. For chrysene
placed by
a 2’-fluoro-2’-deoxyuridine residue (2bOA–
2dOA) again pointed to an important role of backbone
structure at the terminus on the stability of the duplex. We
noted this, but did not pursue 2’-fluoronucleosides them-
Table 2. UV melting points for duplexes of DNA tetradecamers 1 or 2
with the residue of oxolinic acid as the stacking moiety, linked by alkyl
chains
of
different
length,
and
target
RNA
strand
r(AUUAUUAAAAAUUAGUC).
Strand
Melting point [8C][a]
[NaCl]=150 mm [NaCl]=1m
DTm
Hyperchromicity
[%][c]
G
N
[8C][b]
1[d]
21.0Æ0.9
18.5Æ1.0
22.4Æ1.1
23.2Æ1.1
23.2Æ1.1
22.8Æ1.1
22.3Æ0.7
26.4Æ1.0
27.4Æ0.7
26.5Æ1.1
27.0Æ1.0
30.7Æ1.0
31.2Æ0.9
31.3Æ0.8
30.8Æ0.8
31.5Æ0.5
35.1Æ0.8
35.7Æ0.7
–
29
28
30
29
30
33
29
35
35
1aOA
1bOA
1cOA
1dOA
1eOA
2bOA
2cOA
2dOA
0.5
4.2
4.7
4.8
4.3
5.0
8.6
9.2
[a] Average of four melting points at 1 mm strand concentration at the
NaCl concentration given and in sodium phosphate buffer (10 mm, pH 7).
[b] Melting point difference to unmodified control duplex d(TAATTTT-
TAATAAT):r(AUUAUUAAAAAUUAGUC).
[c] Hyperchromicity
upon duplex dissociation at 1m NaCl. [d] Unmodified DNA with control
sequence.
Scheme 4. Synthesis of cap-bearing oligonucleotides with unconventional
linkers.
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Chem. Eur. J. 2011, 17, 11813 – 11827

DNA Probes for Binding RNA Targets
FULL PAPER
Table 3. UV melting points of perfectly matched DNA:RNA duplexes
for the four possible terminal base pairs, with or without a 5’-DNA cap.
as the stacking moiety, either linker led to a melting point
depression in comparison with linker b (1 fCR and 1tCR
versus 1bCR). For other combinations of stacking moieties
and linkers f and t, we also observed no duplex stabilization
(1tAQ, 1tPY, 1 fPE, and 1 fCO). This suggested that we
were still far from a local optimum in linker structure. Since
the base pairs at the termini themselves are similar in
DNA:DNA and DNA:RNA duplexes, and the Tm values
did not come close to those observed for our optimized
DNA:DNA 5’-caps,[19e,21,40] a further “walk” in the structure
space of linkers seemed the most logical step. Furthermore,
it seemed reasonable to construct linkers with at least one
ring structure.
[b]
DNA strand
RNA target strand
Tm [8C][a]
DTm
d
d
d
d
N
r
r
r
r
r
r
r
r
r
r
r
r
G
31.2
30.4
34.8
34.4
39.3
40.3
46.9
44.6
38.5
42.6
47.9
45.7
–
–
–
–
15dOA
16dOA
17dOA
18dOA
15ogOA
16ogOA
17ogOA
18ogOA
+8.1
+9.9
+12.1
+10.2
+7.3
+12.2
+13.1
+11.3
Prior to linker optimization, we asked whether the C5-
linked cap dOA was suitable for stabilizing all of the four
canonical Watson–Crick base pairs (A:U, T:A, C:G, and
G:C). For this, we switched to octamer sequence 5’-
BGGTTGAC-3’, with B=A/C/G/T, partly because we were
necessarily leaving the all-A/T sequence space in this phase,
partly because the melting point of shorter sequences is
more sensitive to small changes in structure, making it
easier to optimize structures. Sequences with any of the four
possible terminal nucleobases were generated (15dOA–
18dOA), starting from supports 19–22 (Scheme 5). These
were reacted with phosphoramidite 5d, and subsequently
acylated with the active ester of oxolinic acid. The data
listed in the middle portion of Table 3 show that strong
melting point increases are induced for each of the possible
terminal base pairs, with DTm values ranging from +8.1 to
+ 12.18C. However, a crucial test for mismatch discrimina-
tion, namely the melting curve for the duplex with a termi-
[a] Average of four melting points at 1 mm strand concentration in sodium
phosphate buffer (10 mm, pH 7) and NaCl (1m). The standard deviation
of four measurements was between Æ0.2 and Æ1.18C. [b] Melting point
difference to the unmodified, perfectly matched control duplex of the
same sequence.
nal A:A mismatch, gave an unsatisfactory result for cap
dOA (Table 4). No more than a marginal increase in the
DTm value compared to the perfectly matched counterpart
was observed, confirming the need for a linker that preorga-
nizes the stacking moiety towards interacting with the cor-
rect terminal bases.
We reasoned that a more rigid linker would prevent the
stabilization of duplexes with anything but the desired
Watson–Crick geometry at the terminus. The most attractive
approach to optimizing the linker was to employ peptide
chemistry. To install a rigidifying ring structure, we based
linkers on l-prolinol. Target strands of the sequences 15–18
were assembled on supports
19–22, through coupling with
phosphoramidite 23, followed
by oxidation to give 24–27
(Scheme 5). The Fmoc-termi-
nated chain was then deprotect-
ed, acylated with the Fmoc-pro-
tected residue of
a second
amino acid, which was then re-
lieved of its Fmoc group, fol-
lowed by coupling of the acti-
vated form of the carboxylic
acid of the stacking moiety
(usually the active ester of oxo-
linic acid).
Scheme 6 shows an overview
of the amino acid residues
tested as parts of the linker.
The list includes d- and l-proli-
nol (o and r, respectively), hy-
droxyprolinol with or without
the bulky terminal DMT group
(h and j, respectively), and a cy-
clohexyl moiety (x). The struc-
tures and syntheses of linker
phosphoramidites 28–30, used
Scheme 5. Representative synthesis of 5’-capped DNA octamers by phosphoramidite coupling with building
block 23, followed by amide-forming couplings with an amino and/or carboxylic acid by using peptide chemis-
try. Fmoc=fluorenylmethyloxycarbonyl; iBu=isobutyryl.
Chem. Eur. J. 2011, 17, 11813 – 11827
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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11821

S. Egetenmeyer and C. Richert
Table 4. UV melting points for duplexes of DNA octamers with the oxo-
linic acid cap linked by different linkers and the perfectly-matched (PM)
target RNA strand rACHTUNGTRENNUNG(GUCAACCU) or target strand rACHUTNGTREN(NGUN GUCAACCA)
with a terminal A:A mismatch next to the molecular cap (MM=mis-
matched).
DNA strand
RNA strand
(GUCAACCU)
RNA strand
rACHTUNGRTENN(UGN GUCAACCA)
r
G
[a]
[b]
[a]
[c]
Tm
[8C]
PM DTm
[8C]
Tm
[8C]
MM DTm
[8C]
d
(AGGTTGAC)
31.2
–
32.0
+0.8
À0.5
À0.2
À4.4
+0.7
À0.9
À0.9
À0.5
À2.0
À1.1
+1.9
+0.5
+0.9
+0.2
À4.9
À4.0
À0.4
À3.7
+4.8
+3.3
–
d(AAGGTTGAC)
15dOA
32.8
39.3
38.5
30.6
31.0
35.1
32.0
36.8
36.5
31.8
31.0
30.8
31.6
38.3
38.3
32.3
38.4
31.0
29.2
+1.6
+8.1
+7.3
À0.6
À0.2
+3.9
+0.8
+5.6
+5.3
+0.6
À0.2
À0.4
+0.4
+7.1
+7.1
+1.1
+7.2
À0.2
À2.0
–
32.3
39.1
34.1
31.3
30.1
34.2
31.5
34.8
35.4
33.7
31.5
31.7
31.8
33.4
34.3
31.9
34.7
35.8
32.5
15ogOA
15opOA
15bpOA
15owOA
15oOA
15ovOA
15ouOA
15oggOA
15osOA
15oyOA
15ozOA
15hgOA
15jgOA
15rOA
15rgOA
15xOA
15xgOA
15xuOA
<15
<15
Scheme 6. Amino acid residues employed in the optimization of the
linker at the 5’-terminus of the DNA chain and stacking moieties that
were based on peptide chemistry. The stacking moieties are shown in
Scheme 2.
[a] Average of four melting points at 1.5 mm strand concentration in
sodium phosphate buffer (10 mm, pH 7) and NaCl (1m). [b] Melting
point difference to unmodified control duplex
(GUCAACCU). [c] Melting point difference to the corresponding modi-
fied perfectly matched duplex.
dACTHNGUTERNNU(G AGGTTGAC):
rACHTUNGTRENNUNG
We also tested the combination of propylene linker b with
proline, but the resulting strand 15bpOA did not show an
increase in Tm. The same was true for prolinol followed by
proline (1opOA), suggesting that the attachment of the
stacking moiety through a cyclic amino acid is disadvanta-
geous. The strands with prolinol and side-chain-bearing a-
amino acid residues, such as valine (1ovOA) or tryptophane
(1owOA), gave DTm values of +5.6 and +3.98C, respective-
ly, for the matched duplex, whereas all three N-substituted
amino acid linkers (15osOA, 15oyOA, and 15ozOA) gave
disappointing results. Apparently, a side chain on the amide
nitrogen of the second residue interferes with the productive
binding of the molecular cap. Probe strands with cyclohexyl
linker x, with or without glycine or b-alanine residue
(1xOA, 1xgOA, and 1xuOA), did not show any duplex-sta-
bilizing effect on the PM duplex, but stabilized MM duplex-
es with a terminal A:A mismatch. When prolinol was re-
placed by hydroxyprolinol (1hgOA) or its DMT-bearing
counterpart (1jgOA), the same effect on the Tm value as for
cap ogOA was measured, both for the PM and the MM
case. This was also largely true for a cap containing d-proli-
nol (1rgOA), which gave a perfect match DTm value of
+7.28C and a slightly decreased mismatch DTm value of
À3.78C. This suggested that the prolinol portion of the
linker, while sensitive to structural changes, tolerates sub-
stituents more readily than the glycine residue in the ogOA
cap.
to introduce linkers o, r, h, j, and x, are shown in the Sup-
porting Information (Figure S2 and Scheme S1 in the Sup-
porting Information). The proximal amino alcohols of the
linkers were combined with amino acids like b-alanine for
increased chain length (u) or a-amino acid residues with (v
and w) or without side chains (g and gg), with an additional
ring structure (p), or with substituents on the backbone ni-
trogen (s, y, and z) to produce a diverse set of linkers.
Table 4 shows the melting points of duplexes of the resulting
probe strands with the complementary RNA target, as well
as the RNA target producing a terminal A:A mismatch. We
assayed duplex stability for this most problematic terminal
mismatch throughout to obtain at least one data point on fi-
delity early on. Compared to a dangling dA residue as the
cap, which gives a DTm value of +1.68C for the perfectly
matched (PM) duplex and a slight decrease in the DTm
value for the terminal A:A mismatch (À0.58C), a number of
caps showed an improvement. From the range of combina-
tions, prolinol with glycine emerged as the most duplex-sta-
bilizing, giving a perfect match DTm value of +7.38C. Nei-
ther a longer linker, as produced by the combination of pro-
linol with b-alanine (1ouOA), nor prolinol with two glycine
units (1oggOA), nor a shorter linker, like prolinol alone
(1oOA or 1rOA), led to greater duplex stabilization than
ogOA, which provided the best combination of affinity and
selectivity (Table 4).
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DNA Probes for Binding RNA Targets
FULL PAPER
Having refined the linker, we then revisited the stacking
moiety one last time. Quinolones other than oxolinic acid
were coupled to the amino-terminal strand featuring the og
linker, followed by deprotection, purification, and melting
curve assays with complementary strands (Table 5). Again,
the most problematic mismatched base combination at the
terminus (A:A) was also assayed. The results in Table 5
show that ogOA itself is the most favorable cap for inducing
both target affinity and base pairing selectivity. Of the other
chinolones tested, only cinoxacine was again found as a pos-
sible replacement for oxolinic acid as the stacking moiety,
although the Tm value was slightly decreased for 15ogCI,
and so was the effect on mismatch discrimination for the ter-
minal A:A mismatch. The remaining residues of chinolones
tested, like ofloxacine, nalidixic acid, and norfloxacine, gave
smaller duplex stabilizing effects (15ogOF, 15ogNA, and
15ogNO, respectively). In order to couple norfloxacine to
the amino terminus of the linker, the secondary amine of
the quinolone was acylated with tert-butylphenoxyacetic
acid (see Scheme S3 in the Supporting Information). Partial
deprotection of the resulting strand during cleavage from
the solid support allowed us to test both the acylated and
non-acylated forms (15ogNT and 15ogNO , respectively).
We also tested more distantly related stacking moieties like
anthraquinone, pyrene, and trimethoxystilbene in combina-
tion with the og linker, but neither 15ogAQ, 15ogPY, nor
15ogMS induced higher melting points than 15ogOA. An
extended version of the ethyl side chain of oxolinic acid (the
phenylethyl group in OB, Scheme 2) was tested by alkylat-
increase in melting point. The non-alkylated species ON was
also tested as a stacking moiety (15ogON). It gave a slightly
decreased duplex melting point compared with 15ogOA.
This suggested that the side chain of OA makes some con-
tacts, but is not critical for the effectiveness of the cap.
Having settled on ogOA as the cap, we tested for stabili-
zation of any of the four canonical terminal Watson–Crick
base pairs (A:U, T:A, C:G, and G:C). For this, we synthe-
sized DNA octamers 15ogOA–18ogOA (Scheme 5) and
measured melting points of the duplexes with RNA target
strands (lower part of Table 3). Compared with the unmodi-
fied DNA:RNA control duplexes, melting point increases of
7.3–13.18C were found. Figure 5 shows an overlay of melting
curves of perfectly matched and mismatch-containing du-
plexes with and without the ogOA cap. The melting point of
the capped, perfectly matched duplex 18ogOA:32 is mas-
sively increased over that of to the unmodified duplex
(31:32, filled symbols), but the duplex with a terminal C:C
mismatch does not experience stabilization by the cap
(31:33 and 18ogOA:33, open symbols).
The improvement in fidelity achieved with the peptidic
linker in ogOA over the alkyl linker in dOA was even more
apparent when the entire matrix of melting points of du-
plexes with terminal mismatches was measured. Figure 6
shows the drop in duplex melting point for a 3’-terminal,
mismatched nucleobase in the RNA strand compared to the
fully matched duplexes. Although either cap improved dis-
crimination for all of the twelve terminal mismatches, com-
pared to the unmodified DNA:RNA duplexes, the effect
was considerably stronger for ogOA in every case. Signifi-
cant selectivity was now observed even for the cases for
which the mismatch gives a more stable duplex than the per-
fect match in the unmodified control duplexes (A:A and
A:G, Figure 1). For the absolute melting points of the entire
4ꢅ4 matrix of base combinations at the terminus, see
Table S4 in the Supporting Information. In the most favor-
ing ethyl-8-hydroxyACTHUNTGRENNUG[1,3]dioxoloACHTUNGTRNE[NUGN 4,5-g]chinoline-7-carboxyl-
ate,[41] and treating the product with sodium hydroxide to
give the carboxylate of OB (see Scheme S2 in the Support-
ing Information). The resulting modified DNA strand
15ogOB showed very similar duplex stability to 15ogOA
when hybridized to its complementary RNA strand, but no
Table 5. UV melting points for duplexes of DNA octamers with different
stacking moieties and the prolinol glycine linker and perfectly matched
target RNA strand rACHTUNGTRENNUNG(GUCAACCU) or target strand rACHUTNGTREN(NGUN GUCAACCA)
producing a terminal A:A mismatch.
DNA strand RNA strand
(GUCAACCU)
RNA strand
rACHTUNGRTENN(UGN GUCAACCA)
r
U
Tm
PM DTm
Tm
MM DTm
[8C][a]
[8C][b]
[8C][a]
[8C][c]
d
(AGGTTGAC)
31.2
38.5
34.0
37.8
34.4
35.6
36.1
38.3
34.7
32.6
37.6
38.1
–
32.0
34.1
35.1
37.4
34.4
32.0
32.1
34.4
34.0
33.1
34.2
33.8
+0.8
À4.4
+1.1
À0.4
0.0
À3.6
À4.0
À3.9
À0.7
+0.5
À3.4
À4.3
15ogOA
15ogOF
15ogAQ
15ogPY
15ogMS
15ogNA
15ogCI
15ogNO
15ogNT
15ogON
15ogOB
+7.3
+2.8
+6.6
+3.2
+4.4
+4.9
+7.1
+3.5
+1.4
+6.4
+6.9
Figure 5. Representative melting curves of duplexes with or without a
molecular cap. Melting curves of octamer DNA:RNA duplexes either
without dACTHUNGTRENNNUG
[a] Average of four melting points at 1.5 mm strand concentration in
sodium phosphate buffer (10 mm, pH 7) and NaCl (1m). [b] Melting
point difference to unmodified control duplex
d
(AGGTTGAC):r-
ing no
rACHTUNGTRENNUNG
~
~
=
^
^
G
r
U
fied perfectly matched duplex.
Chem. Eur. J. 2011, 17, 11813 – 11827
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11823

S. Egetenmeyer and C. Richert
Figure 7. Thermodynamic parameters for DNA:RNA duplex formation
at 378C, as determined from melting curve data by using Meltwin v3.5.[26]
The modified DNA:RNA duplexes feature 15ogOA, 16ogOA, 17ogOA,
and 18ogOA as the probe strands and rACHTUNTRGNEUNG(GUCAACCY), in which Y is
one of the four possible nucleotides, as the target. The columns in the
upper part of the graph show DH at 378C and in the lower part the
values for TDS at 378C are shown. Data from curves detected at 260 nm
from a solution with sodium phosphate buffer (10 mm), NaCl (1m), and
1.5 mm strand concentration. Note the absolute expansion of the y-axis
chosen to highlight differences.
Figure 6. Difference between melting points of two types of capped
DNA:RNA duplexes with terminal mismatches and those of the corre-
sponding perfectly matched duplexes (compare Table 3). The capped
DNA:RNA duplexes consist of the strands 15dOA, 16dOA, 17dOA,
18dOA, 15ogOA, 16ogOA, 17ogOA, 18ogOA, and rACTHNURTGNEU(GN GUCAACCY), in
which Y stands for one of the four nucleosides (A, C, G, or T/U). Melt-
ing curve data was determined from a sodium phosphate buffer (10 mm)
with NaCl (1m) and 1.5 mm strand concentration, detected at 260 nm.
Gratifyingly, cap ogOA also performed well on the tetra-
decamer sequence 1, with a melting point increase of
+9.78C for the duplex of this long all-A/T probe (1ogOA)
with its matched RNA target compared with the unmodified
control (last entry in Table 1). This value compares favor-
ACHTUNGTRENNUNGable cases, for which strong hydrogen bonds and proper
dipole–dipole interactions provide a high level of selectivity,
as for C:G base pairs,[42] the drop in melting point for a mis-
match went up to À20.28C (for the C:C mismatch, com-
pared with the C:G perfect match) in the presence of cap
ogOA. Selectivity thus reached a level similar to that of the
very best caps for DNA:DNA duplexes[19e] or the 3’-terminal
cap of DNA:RNA duplexes published recently.[20]
AHCTUNGTREGaNNUN bly with that of optimized caps for DNA:DNA, such as the
trimethoxystilbene cap,[17] which increases the melting point
of the all-A/T tetradecamer by +5.48C. Finally, and perhaps
most importantly, the cap also fared well when tested as a fi-
delity-enhancing element in a focused hybridization probe
detecting one member of a family of medically relevant
miRNAs, let7a (34),[43,44] versus a closely related miRNA,
let7c (35), for which unmodified DNA does not. Figure 8
shows melting curves for duplexes between DNA probes, 36
and 37, and miRNA, let7c (35) and let7a (34). In the case of
the unmodified, full-length hybridization probe (38), melting
points of duplexes with either target are virtually indistin-
guishable, with a melting-point difference of just 1.88C,
whereas the cap-bearing probe binds selectively, with a DTm
value of +9.28C (for full Tm values, see Table S5 in the Sup-
porting Information).
Further confirmation of the desired effect of the ogOA
cap on DNA:RNA duplexes came from a thermodynamic
analysis of the melting transitions (Figure 7). Absolute
values can be found in Table S10 in the Supporting Informa-
tion. The cap increases the enthalpic gain upon formation
for perfectly matched duplexes by 5.5 to 15.1 kcalmolÀ1,
compared with the unmodified duplexes (Figure 2d). For
mismatch-terminated duplexes, the gain is significantly
smaller. For G:U and G:G mismatch-containing duplexes,
the cap induces a gain of just 2.1–3.4 kcalmolÀ1, and for the
critical A:A base combination, it gives just À5.3 kcalmolÀ1
compared to the unmodified duplex. The enthalpic gains are
accompanied by a significant, but not overwhelming loss of
entropy. The TDS for the capped duplexes decreases, where-
as it remains essentially unchanged for others. In the case of
the terminal C:C mismatch, the decrease in TDS reaches
10.3 kcalmolÀ1 compared with the unmodified duplex, con-
firming a favorable enthalpy–entropy compensation.
Discussion
The termini of duplexes are critical sites for base pairing, as
dissociation of short duplexes typically starts with fraying at
the termini. However, the termini are also the sites of pivo-
tal genetic processes. Replication and transcription occur at
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Chem. Eur. J. 2011, 17, 11813 – 11827

DNA Probes for Binding RNA Targets
FULL PAPER
sorption or micelle formation. Many affinity-increasing ter-
minal substituents known from the literature are fairly lipo-
philic.[34,48] From the results of our optimization it appears
that a level of polarity that is typical for biologically active
compounds, such as peptides and drugs, is well suited for in-
creasing target affinity and base pairing selectivity, even in
challenging cases such as purine–purine pairs at the termi-
nus. The level of rigidity found for the optimized linker in
ogOA seems to be a suitable compromise between flexibility
(needed to adopt the proper binding conformation) and ri-
gidity (needed to suppress too many alternative binding
modes that would stabilize mismatched base pairs and to
prevent too high an entropic cost for binding), at least
within the limited structure space explored with our libra-
ries.
It is also noteworthy that the optimized stacking moiety is
the residue of a quinolone drug. Oxolinic acid is a gyrase in-
hibitor that is used clinically to combat bacterial infections
and has found applications in fields as far from human
health to fish farming.[49] Earlier combinatorial work from
our laboratories, focused on other terminal base pairing ar-
rangements, also identified quinolones as terminal acyl
groups of caps.[22c,40] However, high resolution structures
then revealed that the quinolones disrupted weak base pairs,
rather than stabilizing them, a binding mode readily ration-
alized in the context of their activity as enzyme inhibitors,
but not the desired mode of binding as a cap.[22c] Only when
the terminal nucleoside residue was treated as a linker did a
“composite cap” emerge that does indeed increase affinity
and base pairing selectivity. In our study, we included a li-
brary member that features a nucleosidic linker, but the re-
sulting conjugate was less successful as a high-affinity binder
than other library members. Nevertheless, optimized cap
ogOA does feature a phosphodiester linker, a five-mem-
bered ring, and amide linkages, much like the composite
T*OA cap identified for DNA:DNA duplexes, in which T*
stands for a 5’-amino-5’-deoxythymidine residue with an
amide link to oxolinic acid (OA).[40]
Our results emphasize the importance of the linker. Al-
though the strong propensity of quinolones to stack on nu-
cleobases appears to be a driving force, unless the linker
allows it to do so without disrupting weak base pairs (and
contributes some binding affinity itself), no increase in se-
quence selectivity is imposed upon hybridization probes.
What our study shows more than any previous work is that
not just the length and lipophilicity of the linker is of impor-
tance, but also the conformational rigidity. Only when a ring
structure (prolinol) was introduced, did the OA-terminated
cap increase selectivity at the terminus for the difficult
cases. More rigid structures (prolinol–proline) or the
branched structures tested did not have the same favorable
effect. This suggests to us that the ogOA cap constitutes at
least a local optimum in structure space, in which linker and
stacking moiety both interact favorably with the terminus of
correctly paired strands in DNA:RNA duplexes. The new
cap may aid the detection of biomedically relevant RNA se-
quences.
Figure 8. UV melting curves of duplexes between DNA octamer probes
and two different microRNAs. The unmodified DNA probe (37) or the
cap-bearing octamer probe ogOA (36) were allowed to bind to either of
the targets. Only in the latter case was a significant melting point differ-
ence detected. Conditions: NaCl (1m), sodium phosphate buffer (10 mm),
~
~
^
^
=
DNA (1.5 mm), RNA (1.5 mm). Key: =36:34, =36:35, =37:34,
37:35.
the termini of base paired regions, and translation involves
duplexes with two terminal and one internal base pair (co-
don:anticodon duplexes). Base pairing at the termini is also
fascinating because it occurs in a conformationally less re-
stricted molecular situation in which alternative base pairing
arrangements are more readily realized and refolding or
stacking of blunt ends is common. Therefore, studying base
pairing at the termini is interesting not just for practical ap-
plications. Hybrid DNA:RNA duplexes are a species also
formed during transcription and the formation of primers
during replication. The fundamental interest in the molecu-
lar recognition at termini of DNA:RNA duplexes was one
motivation for our current work that extends earlier work
on DNA:DNA or RNA:RNA duplexes with modified dan-
gling residues or caps.[45]
In the optimization phase of our study, we chose peptidic
linkers (Scheme 1b) both for their flexibility, synthetic ease,
and the modest lipophilicity of the chain. Some long chain
alkanes can stabilize DNA:DNA duplexes,[46] but strongly
lipophilic caps or linkers can lead to hydrophobic dimeriza-
tion,[47] and can complicate purification due to strong ad-
Chem. Eur. J. 2011, 17, 11813 – 11827
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11825

S. Egetenmeyer and C. Richert
pylchlorophosphoramidite (140 mL, 0.6 mmol) was added dropwise at
08C, and the solution was stirred under argon. The ice bath was removed
after 10 min and the solution was allowed to warm to room temperature,
followed by stirring for 2.5 h. The mixture was diluted with CH2Cl2
(5 mL), and washed twice with saturated NaHCO3 solution (5 mL) and
brine (5 mL). The organic layer was dried over MgSO4, and CH2Cl2 was
removed in vacuo. The residue was purified by flash chromatography
(silica, pretreated with eluent containing 2% triethylamine, petroleum
ether/ethyl acetate, 2:1, Rf =0.85) to give 23 as a slightly yellow solid
(126 mg, 0.24 mmol, 81%). 31P NMR (121 MHz, CD3CN): d=147.7 ppm;
1H NMR (300 MHz, CD3CN): d=1.15 (dd, J=10.5 Hz, J=15.8 Hz,
12H), 1.85–1.97 (m, 6H), 3.35–3.82 (m, 6H), 4.18–4.48 (m, 2H), 7.29–
7.86 ppm (m, 8H); MS (ESI): m/z calcd for C29H38N3O4P: 546.25
[M+Na]+; found: 546.25.
Experimental Section
General: Extinction coefficients of the modified oligonucleotides were
calculated as the sum of the extinction coefficients of the unmodified
DNA portion and the extinction coefficient of the stacking moieties at
260 nm given in the literature.[40]
UV melting experiments: Melting curve experiments used sodium phos-
phate buffer (10 mm) at pH 7 with NaCl concentrations of 150 mm or 1m
and strand concentrations of 1.0 mm for duplexes of 1 and 2 or 1.5 mm
(each strand) for duplexes of 15–18. For each sample, at least four curves
from 5 to 808C were measured at a heating or cooling rate of 18CminÀ1
.
Melting points were calculated using Templab 2.0, and the values given
are the average of four curves. The hyperchromicity was calculated as the
difference between the absorption at 808C and that at 58C, divided by
the absorption at 58C.
Analytical data for HPLC-purified modified oligonucleotides and the
MALDI-TOF mass spectra of these compounds are given in the Support-
ing Information.
Thermodynamic data: Thermodynamic data was determined by using
Meltwin, version 3.5 through curve fitting, as described in reference [26].
For each duplex, four melting curves of 38 points were run. The values
given are the average of four results Æ1 standard deviation.
DNA synthesis: DNA oligonucleotides were synthesized on a 1 mmol
scale, following a standard protocol of the manufacturer. Caps were in-
troduced following the general protocols given below, after DMT depro-
tection of the DNA strands on controlled pore glass (cpg). General pro-
tocol A was used for the coupling of phosphoramidite-linker building
blocks to the 5’-terminus of the DNA, followed by Fmoc deprotection[21]
with piperidine in DMF (20%) for 20 min, or MMT deprotection[17] with
TCA deblock solution for DNA synthesis, depending on the linker build-
ing block. After deprotection of the terminal amino group of the linker,
general protocol B was used for peptide coupling of Fmoc-protected
amino acids or carboxylic acids. The oligonucleotides were deprotected
and cleaved off the solid support by treatment with aqueous ammonia
(1 mL, 25%) at 558C for 2 h for oligonucleodides with an all-A/T se-
quence, and for 5 h for oligonucleotides with mixed sequences.[21] The
ammonia was removed by blowing a low stream of nitrogen onto the sur-
face of the solution for 1 h, followed by lyophilization.
Acknowledgements
The authors thank Dr. Amritraj Patra and Dr. Michael Printz for sharing
melting curve data used for thermodynamic analyses, Denis Kusevic, Jo-
hanna Spçrl and Ruth Frank for help with the synthesis of the linker
building blocks, Sebastian Spies for providing oligonucleotides 1bCD,
1bFL, 1bCT, 1bIA, 1bPT, 1bPA, 1bAT, 1bNP, 1bPY, and 1bPQ, Dr.
Eric Kervio for suggesting the use of 2’-fluoro-2’-deoxyuridine, and
Helmut Griesser, Andreas Kaiser, Cora Prestinari, and Heike Vogel for a
review of the manuscript. This work was supported by DFG (grant No.
RI 1063/9–1) and the MINT program of the Ministry for Science Re-
search and the Arts Baden-Wꢂrttemberg (MWK) and febit holding
GmbH (Heidelberg, Germany).
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2 mmol, 60 equiv) were dried together with the DNA-bearing cpg (5 mg,
ca. 0.16 mmol loading) for 2–20 h at 0.1 mbar. After addition of activator
solution (0.25m 4,5-dicyanoimidazol in CH3CN, 40 mL) under argon, the
mixture was agitated at 240 rpm on a vortexer for 2 h. Oxidizer solution
for DNA synthesis (0.02m I2 in pyridine, THF, and water, 100 mL) was
then added, and the mixture was again shaken for 15 min. The superna-
tant was removed, and the cpg was washed five times with acetonitrile
(0.5 mL). Analytical samples were deprotected and cleaved from the cpg
and evaluated by MALDI-TOF MS.
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FULL PAPER
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Received: June 15, 2011
Published online: September 20, 2011
Chem. Eur. J. 2011, 17, 11813 – 11827
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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