Unusual RNA and DNA binding properties of a novel pyrrolidine-amide oligonucleotide mimic (POM)

Article abstract of DOI:10.1039/b006903p

A pentameric thymidyl pyrrolidine-amide oligonucleotide mimic (POM) was synthesised and shown to bind with very high affinity to complementary single stranded RNA and DNA, whilst exhibiting kinetic binding selectivity for RNA over DNA.

Full text of DOI:10.1039/b006903p

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Unusual RNA and DNA binding properties of a novel pyrrolidine–amide
oligonucleotide mimic (POM)†
David T. Hickman,a Paul M. King,b Matthew A. Cooper,c Jonathan M. Slaterb and Jason Micklefield*a
a Department of Chemistry, UMIST, Faraday Building, PO Box 88, Manchester, UK M60 1QD.
E-mail: jason.mickelfield@umist.ac.uk
b Department of Chemistry, Birkbeck College, University of London, 29 Gordon Square, London, UK WC1H 0PP
c
University Chemical Laboratory, Lensfield Road, Cambridge, UK CB2 1EW
Received (in Cambridge, UK) 23rd August 2000, Accepted 16th October 2000
First published as an Advance Article on the web
A pentameric thymidyl pyrrolidine–amide oligonucleotide
mimic (POM) was synthesised and shown to bind with very
high affinity to complementary single stranded RNA and
DNA, whilst exhibiting kinetic binding selectivity for RNA
over DNA.
The sugar–phosphodiester backbone of nucleic acids has been
replaced by many alternative neutral and anionic backbones.1
There have also been reports of zwitterionic and cationic
oligonucleotides with pendant aminoalkyl side chains attached
to the sugar ring, pyrimidine base, or to various phosphorus
internucleoside linkages.2 Despite this, only a few de novo
modified oligonucleotides have been reported with positively
charged backbones.3 Here we introduce a novel pyrrolidine–
amide oligonucleotide mimic (POM) 1 (Scheme 1), which is
derived by replacing the furanose sugar of native nucleic acids
with a pyrrolidine ring which will be protonated and positively
charged at physiological pH.3d An X-ray crystal structure4 of a
protonated pyrrolidine ring, which is stereochemically identical
and both electronically and sterically similar to the pyrrolidine
ring in 1, closely resembles the northern (N) conformation of
Scheme 2. Reagents and conditions: i, LiBH4 (2 eq.), THF, 0 °C ? rt, 15
uridine in the crystalline state.5 Semi-empirical quantum
h, 69%; ii, phthalimide, PPh3, DEAD (all 1.3 eq.) in THF, 215 °C ? rt, 15
mechanical calculations (MOPAC 6.0) also revealed that the
h, 63%; iii, 25–30% aq. MeNH2, 1 h, 40 °C, 89%; iv, bromoacetic anhydride
lowest energy conformation of a model pyrrolidine 2 closely
(1 eq.), AcCN–CH2Cl2, 28 °C ? rt, 5 min, 97%; v, CH2Cl2–CF3CO2H
resembles the preferred N-conformation of the ribose ring in
native RNA. In addition, the rigid amide linkage is a viable
replacement for the phosphodiester group in DNA resulting in
modified oligonucleotides that form stable duplexes with RNA
and DNA.6
(2+1), 4 h, 86%; vi, tert-butyl bromoacetate (1.5 eq.), DIPEA (3 eq.), DMF,
0 °C ? rt, 18 h, 92%; vii, CH2Cl2–CF3CO2H (4+1), 3 h, then pyridine (2
eq.), CF3CO2Pfp (1.2 eq.), DMF, 2 h, 78%; viii, 8 + 9 (1+1), DIPEA (3 eq.),
DMF, rt, 18 h, 98%; ix, 7 + 11 (1+1) CH2Cl2, rt, 3 h, 100%; x, CH2Cl2–
CF3CO2H (4+1), rt, 4 h, then DIPEA (5 eq.), DMF, 9, rt, 97%; xi and xii,
repeat conditions for 12 ? 13, 96 and 91%; xiii, MeOH–H2O (1+1)
saturated with HCl(g), rt, 2 h, 95%. DIPEA = diisopropylethylamine, Pfp =
pentafluorophenyl, Phth = phthalimide, T = thymidyl. New compounds
were characterised by 1H and 13C NMR, IR, UV, MS, mp, [a]D.
To begin investigating the nucleic acid binding properties of
POM a pentamer, T5-POM 3, was synthesised, in solution, from
trans-4-hydroxy-
-proline via the ester 47 (Scheme 2). Lithium
L
borohydride reduction of the ester and benzoyl groups of 4 gave
the alcohol 5, which was subjected to a Mitsunobu reaction to
afford the phthalimide derivative 6. Removal of the phthalimide
and Boc groups gave the amines 7 and 8 respectively, which
were used as the building blocks for the construction of
oligomers by an N-alkylation or N-acylation strategy. In the
former approach primary amine 7 was treated with bromoacetic
anhydride to give the bromoacetamide 9 which was coupled
with the secondary amine 8 resulting in the dimer 12. Boc
deprotection of 12 and a second coupling with 9 results in the
trimer 13. These steps were repeated to give the tetramer 14 and
pentamer 15, which on treatment with HCl resulted in T5-POM
3 as an highly water soluble HCl salt. Alternatively, treatment of
the secondary amine 8 with tert-butyl bromoacetate followed by
acidolysis and esterification with pentafluorophenyl trifluoro-
acetate gave the Pfp-ester 11 which was used to acylate primary
amine 7 to give dimer 12.
UV thermal denaturation experiments were then carried out
with an equimolar mixture of T5-POM 3 and poly(rA) (Table 1).
At pH 7, 0.12 M K+ a single hyperchromic shift was observed
with a melting temperature (Tm) of 49 °C (ca. 10 °C per base).
In comparison, native d(T)5 showed no hyperchromic shift with
poly(rA), above 8 °C under identical conditions, whilst d(T)20
formed a duplex with poly(rA) with a Tm of 42 °C (2.1 °C per
base). Peptide nucleic acid (PNA) lys-T5-lysNH2 exhibited only
slightly higher affinity for poly(rA) (Tm = 56 °C). Furthermore,
no hyperchromic shifts were observed for T5-POM 3 with non-
complementary poly(rC), (rG) and (rU), whilst Job plots of 3
with poly(rA) revealed a 1+1 binding stoichiometry consistent
with the formation of a Watson–Crick base paired duplex.
Scheme 1
† Electronic supplementary information (ESI) available: UV thermal
denaturation curves, Job plots and SPR sensograms for TS-POM 3 binding
to DNA and RNA. See http://www.rsc.org/suppdata/cc/b0/b006903p/
DOI: 10.1039/b006903p
Chem. Commun., 2000, 2251–2252
This journal is © The Royal Society of Chemistry 2000
2251

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Table 1 Transition melting temperatures (Tm) of T5-POM 3 with poly(rA)
and poly(dA).
Tm/°C
[K+]/M
pH
Poly(rA)a
Poly(dA)b
0.12
0.22
0.62
1.20
0.12
0.12
0.12
0.12
7.0
7.0
7.0
7.0
8.0
7.5
6.5
6.0
49 (56)c
52
54
55
45
46
54
57
57 (48)c
n.d.d
42, 66e
61
n.d.d
n.d.
n.d.
35, 64e
a T5-POM 3 and poly(rA) (42 mM each in bases) in 10 mM K2HPO4(total
volume 1 cm3) adjusted to the appropriate ionic strength and pH. UV
absorbance (A260) was recorded with heating at 5 °C min21 from 25 to
93 °C, cooling at 0.2 °C min21 to 15 °C and heating at 0.2 °C min21 to 93
°C. The Tm was determined from the first derivative of the final slow heating
curve. b 3 and poly(dA) (210 mM each in bases) in 10 mM K2HPO4 (total
volume 0.2 cm3) were incubated for 48–96 h at 25 °C, diluted to 1 cm3
adjusting to the appropriate ionic strength and pH, cooled to 15 °C at 1 °C
min21, heated at 0.2 °C min21 to 93 °C from which the Tm was measured
as above. c Tm values for lys-T5-lysNH2 PNA (PE Biosystems). d Tm not
determined. e Two transitions observed.
Fig. 1 Normalised UV absorbance (A260) of T5-POM 3 with poly(rA) and
(dA) vs. time at 25 °C. 3 and poly(dA) (42 mM each in bases), 0.12 M K+,
pH 7 (2); 3 and poly(dA) (210 mM), 0.12 M K+, pH 7 (8); 3 and poly(rA)
(42 mM), 0.22 M K+, pH 7 (:) ; 3 and poly(rA) (42 mM), 0.12 M K+, pH 7
(5); 3 and poly(rA) (42 mM), 0.12 M K+, pH 6 (-).
was then injected across each surface and the SPR response was
measured against time (see ESI†). This revealed that 3 does bind
strongly to both d(A)20 and r(A)20 but associates faster with
r(A)20 than d(A)20. Significantly, the response sensogram of the
mixed sequence DNA was identical to the control non–
derivatised surface.
Surprisingly however, increasing the salt concentration resulted
in slightly higher Tm values. This is in contrast to other cationic
modified oligonucleotides that show a marked decrease in
duplex and triplex stability with RNA and DNA at higher salt
concentration, which is attributed to a reduction in the
electrostatic attraction between the oppositely charged back-
bones.3 The Tm of 3 with poly(rA) was also highly dependent on
pH with more stable duplexes formed at lower pH. This
suggests that the extent of protonation of the nitrogen atom of
the pyrrolidine ring, is important for binding to RNA. However,
factors other than electrostatic attraction, perhaps conforma-
tional changes brought about by protonation, are more likely to
be the cause of increased duplex stability.
Remarkably no melting was observed between T5-POM 3
and equimolar poly(dA) under identical conditions. Only after a
five-fold increase in concentration of both 3 and poly(dA)
followed by an extended period of incubation (48–96 h) was it
possible to observe melting, suggesting T5-POM binds much
more slowly to poly(dA) than poly(rA). On the other hand the
affinity of 3 for poly(dA) was considerably higher than for
poly(rA) (Tm = 57 °C, pH 7, 0.12 M K+), whilst lys-T5-lysNH2
PNA exhibited a lower affinity for poly(dA). Noticeably upon
increasing the salt concentration (0.62 M K+) or lowering the
pH to 6, two melting temperatures were observed consistent
with triple helix to duplex and duplex to single strand
transitions. Job plots of 3 with poly(dA) indicated a 2+1 (T+A)
binding stoichiometry consistent of triplex formation.
In conclusion we have introduced a novel class of modified
nucleic acids with a pyrrolidine–amide backbone and shown
that the pentamer T5-POM 3 binds sequence specifically to both
ssDNA and ssRNA with an affinity that is much higher than
native nucleic acids. Furthermore, T5-POM binds much faster to
ssRNA than ssDNA. Other oligonucleotides such as 2A,5A-linked
RNA and DNA exhibit a thermodynamic binding selectivity for
native ssRNA over ssDNA,8 but as far as we are aware T5-POM
is the first modified oligonucleotide that can kinetically
discriminate between the two. This kinetic preference may be
due to folding of the polyadenylates induced by base pairing
with T5-POM, given that RNA would be expected to fold more
readily than DNA. In addition the formation of tertiary
interactions could also explain the high stability of T5-POM
complexes with complementary nucleic acids. The synthesis of
longer mixed sequence POMs, using solid phase methods, is
underway in order to explore the generality of these findings.
We thank the EPSRC for a studentship to D. T. H.
Notes and references
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To investigate the difference in the association kinetics of T5-
POM 3 with DNA and RNA, the change in A260 with time was
recorded immediately following mixing of equimolar amounts
of the polyadenylates with 3 (Fig. 1). With poly(rA) at pH 7,
0.12 M K+ and a base concentration of 42 mM for each oligomer,
a 29% hypochromic shift was observed with a t1/2 for
association of ca. 7 min. Under identical conditions no
hypochromic shift was observed with poly(dA) even after 15 h.
However, increasing the concentration of both T5-POM 3 and
poly(dA) fivefold resulted in a moderate 6% hypochromic shift
with a t1/2 of at least 30 min. This clearly shows that T5-POM 3
binds much more slowly to poly(dA) than (rA). It was also
apparent from these experiments that T5-POM binds faster to
poly(rA) at lower pH and salt concentration, suggesting that
electrostatic attraction increases the rate of association.
The high affinity, sequence specific binding and relative rates
of association of T5-POM 3 with DNA and RNA were
confirmed using surface plasmon resonance (SPR). In these
experiments 5A-biotinylated d(A)20, r(A)20 and a mixed se-
quence DNA 30–mer were immobilised via streptavidin into a
dextran matrix upon a gold surface. A solution of T5-POM 3
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