Triple helical recognition of pyrimidine inversions in polypurine tracts of RNA by nucleobase-modified PNA

Article abstract of DOI:10.1039/c1cc14706d

Peptide nucleic acids containing 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) heterocycles recognized C-G and U-A inversions in a polypurine tract of double helical RNA with high affinity and sequence selectivity at pH 6.25. E-modified PNA bound

Full text of DOI:10.1039/c1cc14706d

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Triple helical recognition of pyrimidine inversions in polypurine tracts of
RNA by nucleobase-modified PNAw
Pankaj Gupta,z Thomas Zengeyaz and Eriks Rozners*
Received 31st July 2011, Accepted 24th August 2011
DOI: 10.1039/c1cc14706d
Peptide nucleic acids containing 2-pyrimidinone (P) and 3-oxo-
2,3-dihydropyridazine (E) heterocycles recognized C–G and
U–A inversions in a polypurine tract of double helical RNA
with high affinity and sequence selectivity at pH 6.25. E-modified
PNA bound strongly to bacterial A-site RNA, while no binding
was observed to the human A-site RNA.
common triple helical recognition. Moreover, the requirement
for cytosine (pKa = 4.5) protonation greatly decreases the
stability of C+*G–C at physiological pH. We recently
reported6 that the problem of low affinity could be overcome
by using peptide nucleic acid (PNA), a neutral oligonucleotide
analogue that does not suffer from the electrostatic repulsion.
Although binding of PNA to double helical DNA has been
extensively explored,7 triple helix formation between PNA and
double helical RNA was unknown before our study.6 This
discovery inspired a hypothesis that PNA may serve as a
general ligand for sequence selective recognition of biologically
relevant RNA helices.
Despite significant research activity,8 the requirement for
long purine tracts remains a major limitation of triple helical
recognition. Biologically relevant double-helical RNAs typically
do not contain long polypurine stretches. However, it is common
to find seven or more contiguous purines interrupted by one or
two pyrimidines in ribosomal RNAs9 and microRNAs.10
Thus, if the sequence range of triple helical recognition could
be expanded to recognize isolated pyrimidines, the approach
could be rendered useful for fundamental studies in RNA
biology as well as practical biomedical applications.
Recent discoveries of important roles that non-coding RNAs
play in regulating gene expression make them attractive
targets for molecular recognition.1 However, most non-coding
RNAs are in a double helical conformation and molecular
recognition of such structures is a formidable problem.
Designing small molecules that selectively recognize RNA
using hydrophobic or electrostatic interactions has been a
challenging and involved process.2 Herein, we show that
RNA can be recognized with high affinity and sequence
selectivity using nucleobase-modified PNA that forms stable
triple helix in the major groove of RNA duplex.
Hydrogen bonding is an inherently effective means of
selective recognition of nucleic acids. Dervan and co-workers
pioneered this concept in DNA recognition using minor
groove-binding polyamides3 and major groove-binding oligo-
nucleotides.4 Compared to DNA, the minor groove of RNA is
wide and shallow and less suited for molecular recognition. The
major groove of RNA is deep and narrow, which complicates
triple helical binding. RNA triple helices are little studied.
Relatively stable RNA triple helices are formed via parallel
binding of a pyrimidine oligoribonucleotide to a purine tract of
the double helical RNA.5 The sequence selectivity is achieved
through Hoogsteen hydrogen bonding, which allows formation
of U*A–U and C+*G–C triplets (Fig. 1).
We recently found that 5-methylisocytidine when installed
in PNA recognized the C–G inversion in polypurine tract of
RNA with slightly higher affinity than the natural nucleobases,
though the sequence selectivity of recognition was low.11 Herein
we show that 2-pyrimidinone (P) and 3-oxo-2,3-dihydro-
pyridazine (E) recognized C–G and U–A inversions (Fig. 1)
with high affinity and sequence selectivity at pH 6.25. The use
of P base in PNA had no precedent before our study, although
derivatives of pyrimidinone have been used as oligonucleotide
modifications for recognition of C–G inversions in polypurine
Practical applications of triple helical recognition of nucleic
acids are limited by (1) low affinity of the third strand oligo-
nucleotide for the double helix caused, at least in part, by
electrostatic repulsion between the negatively charged phos-
phate backbones and (2) the requirement for long homopurine
tracts, as only U*A–U and C+*G–C triplets are used in the
Department of Chemistry, Binghamton University,
The State University of New York, Binghamton, New York 13902,
USA. E-mail: erozners@binghamton.edu; Tel: (+) 1-607-777-2441
w Electronic supplementary information (ESI) available: Details of
PNA synthesis, ITC experiments and data, UV melting data and
copies of NMR spectra. See DOI: 10.1039/c1cc14706d
z These authors contributed equally to this work.
Fig. 1 Hoogsteen triplets recognition of purines and pyrimidines.
Chem. Commun., 2011, 47, 11125–11127 11125
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Scheme 1 Synthesis of P and Pex PNA monomers. Steps: (a) EDC,
DhbtOH, DMF, 40 1C, 12 h, 3a 67%, 3b 65%; (b) Pd/C, H2, MeOH or
MeOH:CH2Cl2 (1 : 1), rt, 3h, 4a 91%, 4b 84%.
tracts of DNA.12 The E base was originally designed as a PNA
modification by Nielsen and co-workers,13 who used it to
recognize T–A inversions in DNA. Apart from our preliminary
study using 5-methylisocytidine,11 recognition of pyrimidine
inversions in polypurine tracts of double helical RNA had no
previous precedents.
Fig. 2 Sequences of RNA hairpins and PNA ligands.
hypothesis that an extended linker would improve binding
affinity, PNA4 featuring the Pex base (monomer 4b) had
significantly higher affinity than PNA3 for the matched target
HRP3 (cf., Table 1, entries 3 and 4). However, the higher
affinity was accompanied by somewhat lower sequence selectivity
of PNA4. Surprisingly, PNA5 featuring the E base had high
binding affinity to all hairpins tested without any sequence
selectivity. This result was in contrast to the original report13
that E base demonstrated high selectivity for T and U over
other nucleobases in DNA at pH 7. The discrepancy prompted
us to investigate the binding of modified PNAs at higher and
more physiologically relevant pH.
Our study started with synthesis of Fmoc-protected P, Pex and
E PNA monomers 4a, 4b and 7, respectively (Scheme 1 and 2).
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide (EDC) and
3-hydroxy-1,2,3-benzotriazine-4(3H)-one (DhbtOH) mediated
coupling of the commercially available (2-oxo-1(2H)-pyrimidinyl)-
acetic acid 2a and PNA backbone 1 followed by hydrogenation
gave P PNA monomer 4a.
Analogous N,N0-dicyclohexylcarbodiimide (DCC) mediated
coupling of 1 with the known carboxylic acid 513 followed by
hydrogenation gave E PNA monomer 7 (Scheme 2). Inspired by
the design of the E base, which features longer linker between
the backbone of PNA and the heterocycle, we also prepared an
extended variant of P, Pex PNA monomer 4b (Scheme 1). Our
hypothesis was that the one carbon longer linker in 4b would
relax the conformation of PNA’s backbone allowing better
positioning of the P base for recognition of the C-G inversion.
Monomers 4a, 4b and 7 were used in a standard PNA
synthesis protocol on an Expedite 8909 DNA synthesizer to
prepare modified PNA nonamers PNA3, PNA4 and PNA5,
respectively (Fig. 2). The target RNA hairpins HRP1-HRP4
(containing a variable base pair) were chosen from our previous
studies.6,11 We expected that the modified PNAs would form the
following matched triplexes: the P-modified PNA3 and PNA4
with HRP3 and the E-modified PNA5 with HRP4. The binding
affinity and sequence selectivity were studied using isothermal
titration calorimetry (ITC), as previously described by us.6
ITC results (Table 1, Fig. S1–19w) showed that at pH 5.5 the
unmodified PNA1 and PNA2 had excellent binding affinity
(Ka 4 8 ꢀ 108) and good selectivity for the matched hairpins
HRP1 and HRP2, respectively. Replacement of the variable
base in PNA with the P base (monomer 4a) led to an overall
decrease in binding affinity for PNA3, while good binding
selectivity was maintained (Table 1, entry 3). In support of our
Initial ITC experiments did not detect binding of the
unmodified PNA2 to HRP2 at pH 7 (Ka o 103). However,
good affinity was observed at pH 6.25 (Table 2, Fig. S20–35w),
which has biological relevance being in the range of cytoplasmic
pH of acidophilic bacteria.14 Consistent with unfavourable
cytosine (pKa = 4.5) protonation, changing the pH from 5.5
to 6.25 reduced the affinity of PNAs, while the sequence
selectivity was significantly improved (cf., Tables 1 and 2).
The increase of pH restored the expected binding profile for
PNA5, which now had the highest affinity for the matched
HRP4 (bold in entry 4, Table 2). It is conceivable that lower
pH may have favored protonation or alternative tautomeric
forms of E, which may explain the high affinity and low
selectivity of PNA5 at pH 5.5. Most importantly, at pH 6.25
the affinity of PNAs targeting single isolated pyrimidine
interruption was similar to the affinity of unmodified PNAs
targeting all-purine strands (see bold numbers in Table 2). As
observed in our previous study,6 the binding stoichiometry
(Table S1) was consistent with a 1 : 1 PNA-RNA triple helix.
The P and E bases showed excellent sequence selectivity for
the target C–G and U–A base pairs, respectively.
The binding of PNAs to RNA hairpins was further con-
firmed by the expected biphasic UV melting curves typical for
triple helices (Fig. 3, for full set of data, see Fig. S37–40w). The
low temperature transition was assigned to the dissociation of
Table 1 Binding of PNA to RNA hairpins at pH 5.5a
HRP1
(G–C)
HRP2
(A–U)
HRP3
(C–G)
HRP4
(U–A)
Entry
PNA (variable base)
1
2
3
4
5
PNA1 (C)
PNA2 (T)
PNA3 (P)
PNA4 (Pex
PNA5 (E)
890
72
9
47
96
820
3
172
210
74
62
46
293
NDb
3
8
3
6
38
)
270
Scheme 2 Synthesis of E PNA monomer. Steps: (a) DhbtOH, DCC,
DMF, 0 1C, 1h then rt, overnight, 89%; (b) Pd/C, H2, ethanol, rt, 3h,
73%.
a
b
Association constants Ka ꢀ 106 Mꢁ1 in sodium acetate buffer. Not
determined.
c
11126 Chem. Commun., 2011, 47, 11125–11127
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Table 2 Binding of PNA to RNA hairpins at pH 6.25a
HRP1
(G–C)
HRP2
(A–U)
HRP3
(C–G)
HRP4
(U–A)
Entry
PNA (variable base)
1
2
3
4
PNA1 (C)
PNA2 (T)
PNA4 (Pex
PNA5 (E)
8.1
7.5
LBb
3.0
LBb
20.0
LBb
0.5
LBb
LBb
4.4
LBb
0.9
)
LBb
28.0
LBb
a
Association constants Ka ꢀ 106 Mꢁ1 in sodium acetate buffer.
Ka estimated o 0.01; the low binding prevented accurate curve fit.
b
the PNA from the triple helical structure. The high temperature
transition was assigned to the melting of the hairpin to single
strand. The triple helix melting for the matched PNA4-HRP3
(tm = 43 1C, solid line in Fig. 3) was shifted 14 1C higher than
the transitions of the mismatched complexes (tm = 19 to 29 1C).
Overall, the melting data were consistent with triple helix
formation and confirmed our ITC results.
Fig. 4 Binding of E-modified PNA6 to models of bacterial (HRP5)
and human (HRP6) A-site RNAs.
In summary, PNA nonamers modified with P and E nucleo-
bases recognized single isolated pyrimidine interruptions in
polypurine tracts of double helical RNA with similar affinity
and sequence selectivity than unmodified PNAs binding to all-
purine strands of RNA at pH 6.25. Preliminary results suggested
that the approach could be further developed to recognize
complex biologically relevant RNAs featuring bulges and non-
canonical base pairs, such as pre-microRNAs and ribosomal
RNAs.9,10 Further development of more basic cytosine analogues
should increase the binding affinity at physiological conditions.
Although recognition of pre-microRNA is a relatively new
area of research, promising results have already been reported
that binding of helix-threading peptides inhibit maturation of
pre-microRNA.15
The encouraging results obtained in our model system (Fig. 2)
prompted us to check if nucleobase-modified PNAs could
recognize purine-rich strands in biologically important RNAs.
Intriguingly, the sequence of ribosomal A-site conserved
among several pathogenic bacteria, such as E. coli, P. aeruginosa
and S. aureus, features a stretch of eight purines (Fig. 4, bold
in HRP5) interrupted by single uridine.9 ITC experiments
(Fig. S36w) showed that the E-modified PNA6 recognized
the bacterial A-site with affinity similar to that observed in
our model hairpins. The binding stoichiometry was close to
1 : 1, as expected for the triple helix. In contrast, we observed
no binding of PNA6 to HRP6, which features the sequence of
the human ribosomal A-site.
We thank Binghamton University and NIH (R01 GM071461)
for financial support of this research.
Notes and references
Remarkably, the non-canonical A*G and A*A base pairs
and the looped out adenosine did not significantly lower
the stability of the PNA-RNA complex. We expected that of
PNA6 would show excellent selectivity for HRP5 over HRP6
because the purine-rich strands of human and bacterial A-site
sequences had only four out of nine nucleosides common (bold
in HRP6). This is in contrast to the A-rich loop, the target of
aminoglycoside antibiotics, which is remarkably similar for
different organisms.
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Fig. 3 UV thermal melting curves of Pex-modified PNA4 bound to
HRP1-HRP4. Solid line is the matched PNA4-HRP3 complex.
c
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Chem. Commun., 2011, 47, 11125–11127 11127