Triple-helical recognition of RNA using 2-aminopyridine-modified PNA at physiologically relevant conditions

Article abstract of DOI:10.1002/anie.201207925

Peptide nucleic acids containing thymidine and 2-aminopyridine (M) nucleobases form stable and sequence-selective triple helices with double-stranded RNA at physiologically relevant conditions. The M-modified PNA showed unique RNA selectivity by having tw

Full text of DOI:10.1002/anie.201207925

Angewandte
Chemie
DOI: 10.1002/anie.201207925
RNA Recognition
Triple-Helical Recognition of RNA Using 2-Aminopyridine-Modified
PNA at Physiologically Relevant Conditions**
Thomas Zengeya, Pankaj Gupta, and Eriks Rozners*
Compared to DNA, molecular recognition of double-
stranded RNA has received relatively little attention. Until
the early 1990s, RNA was viewed as a passive messenger in
the transfer of genetic information from DNA to proteins.
However, since the discovery that RNA can catalyze chemical
reactions, the number and variety of non-coding RNAs and
the important roles they play in biology have been growing
steadily.[1] Currently, the functional importance of most RNA
transcripts is still unknown, and it is fairly safe to predict that
we will discover many more regulatory RNAs in the near
future. The ability to selectively recognize and control the
function of such RNAs will be highly useful for both
Figure 1. Standard and modified Hoogsteen triplets.
fundamental research and practical applications. However,
recognition of double-helical RNA by sequence-selective
ligand binding is a formidable challenge.[2,3]
selective recognition of the RNA duplex can be achieved at
physiologically relevant conditions by replacing cytosine with
a more basic (pKa = 6.7[9]) heterocycle, 2-aminopyridine
(abbreviated as M in Figure 1).
Povsic and Dervan pioneered the chemical modulation of
the cytosine pKa by showing that triple helices containing 5-
methylcytosine were more stable at higher pH than those of
unmodified DNA.[10] More recently, derivatives of 2-amino-
pyridine have been used to increase the stability of DNA
triple helices at high pH.[11–14] Before our studies, this
approach had not been used in PNA.
An alternative approach has used neutral nucleobases
that mimic the hydrogen-bonding pattern of protonated
cytosine. The most notable examples are pseudoisocytosine
(abbreviated as J in Figure 1) by Kan and co-workers,[15]
methyloxocytosine by McLaughlin and co-workers,[16,17] and
a pyrazine derivative by von Krosigk and Benner.[18] The J
base is widely used in PNA to alleviate the pH dependency of
PNA-DNA triplexes.[19,20] The use of modified heterocycles to
recognize double stranded RNA at physiologically relevant
conditions had no precedent before our studies.
We started by comparing the binding of unmodified
PNA1, J-modified PNA2 and M-modified PNA3 to HRPA
(Figure 2), an A-rich RNA hairpin similar to the model
systems used previously by us.[4,5] Fmoc-protected J monomer
1 was synthesized according to the literature procedure.[21]
The M monomer 2 was synthesized from Fmoc-protected
PNA backbone 3 and the known carboxylic acid 4 using DCC
mediated coupling followed by deprotection of the allyl group
as previously described by us (Scheme 1).[5] All of the PNAs
were synthesized using a standard PNA procedure on an
Expedite 8909 DNA synthesizer, purified by HPLC, and
characterized by mass spectrometry as previously
reported.[4–6]
Recently, we proposed that biologically relevant double-
helical RNAs could be recognized by major-groove triple-
helix formation using peptide nucleic acid (PNA).[4–6] We
showed that PNAs as short as hexamers formed stable and
sequence selective Hoogsteen triple helices with RNA
duplexes (Ka > 107 mÀ1) at pH 5.5.[4] A limitation of triple
helical recognition was the requirement for long homopurine
tracts, as only the Hoogsteen T(U)*A-T(U) and C + *G-C
triplets could be used (Figure 1). We found that modification
of PNA with 2-pyrimidinone[7] and 3-oxo-2,3-dihydropyrida-
zine (E)[8] nucleobases allowed efficient and selective recog-
nition of isolated C-G and U-A inversions, respectively, in
polypurine tracts of double helical RNA at pH 6.25.[5,6]
However, the high affinity of PNA at pH 5.5 was greatly
reduced at pH 6.25, and no binding could be observed at
physiologically relevant salt and pH 7.4.[5] The remaining
problem was the unfavorable protonation of cytosine, which
was required for formation of the Hoogsteen C + *G-C
triplets (Figure 1). Because its pKa = 4.5, cytosine is hardly
protonated under physiological pH, which greatly decreases
the stability of the triple helix. Herein we provide an efficient
solution to this problem and demonstrate that sequence
[*] T. Zengeya, Dr. P. Gupta, Dr. E. Rozners
Department of Chemistry, Binghamton University
The State University of New York
Binghamton, NY 13902 (USA)
E-mail: erozners@binghamton.edu
Homepage: http://www2.binghamton.edu/chemistry/people/roz-
ners/rozners.html
[**] We thank Binghamton University and the NIH (R01 GM071461) for
support of this research. The Regional NMR Facility (600 MHz
instrument) at Binghamton University is supported by the NSF
(CHE-0922815).
Following the same approach as in our previous stud-
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201207925.
ies,[4–6] we used isothermal titration calorimetry (ITC) and UV
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reported that the affinity of an unmodified PNA 15 mer
(having 5 isolated cytosines) for a DNA duplex dropped by
three orders of magnitude (Kd changed from 2 nm to 2.2 mm)
when changing the pH from 5.5 to 7.2. Substitution of all five
cytosines by J base increased the affinity only about tenfold
(Kd = 0.15 mm).[20] Thus, our result was qualitatively consis-
tent with that reported by Nielsen, only smaller in magnitude,
and suggested that the positive charge on cytosine contributed
significantly to stability of the Hoogsteen triplet, presumably
by electrostatic attraction to the negatively charged nucleic
acid. Consequently, an ideal design for recognition of G-C
pairs would include both a correct hydrogen bonding pattern
and a positive charge on the heterocycle. Because unmodified
PNA containing cytosine (pKa = 4.5) forms a stable triple
helix at pH 5.5, we hypothesized that PNA modified with 2-
aminopyridine M (pKa = 6.7) would form at least equally
strong triple helices at physiological pH 7.4 (owing to a similar
pH/pKa difference).
Figure 2. RNA, PNA, and DNA sequences used to compare C, J, and
M nucleobases.
Confirming our hypothesis, ITC showed that M modifi-
cation strongly enhanced the binding affinity of PNA3. In
acetate buffer at pH 7, M-modified PNA3 had about two
orders of magnitude higher affinity (Ka = 3.7 ꢀ 108) for HRPA
than the J-modified PNA2 (Figure 2 and Table 1). Under
physiologically relevant conditions, PNA3 bound to HRPA
with Ka = 1.8 ꢀ 107, which was an order of magnitude higher
than the affinity of PNA2 at the same conditions and twice
that of unmodified PNA1 at pH 5.5. The larger drop in
affinity for PNA3 compared to PNA2 going from acetate to
phosphate buffer is most likely due to screening of the
electrostatic interactions (that are more important for the
charged M) by higher salt concentration and the presence of
MgCl2 in the physiologically relevant buffer. Most remark-
ably, binding of PNA3 to the matched DNA hairpin (DNAA)
in physiological phosphate buffer was about two orders of
magnitude weaker (Ka = 3 ꢀ 105) than binding to RNA
HRPA. This result suggested that the M-modified PNA
might have unique selectivity for triple helical recognition of
RNA over DNA. For all experiments at physiologically
relevant conditions, fitting of ITC titration curves gave a 1:1
Scheme 1. Synthesis of M PNA monomer: a) DCC, 3-hydroxy-1,2,3-
benzotriazin-4(3H)-one, DMF, RT, overnight, 57%; b) [Pd(PPh3)4], N-
ethylaniline, THF, RT, 2 h, 79%.
thermal melting to characterize the binding of PNA to RNA
hairpins. ITC directly measures the enthalpy of binding and,
by fitting the binding data, provides binding affinity (associ-
ation constant Ka in mÀ1) and stoichiometry (the ratio of PNA
to RNA in the final complex).[22] Owing to its operational
simplicity, reliability, and rich thermodynamic data, ITC is
one of the best methods to study ligand binding to RNA. The
unmodified PNA1 formed a stable triplex with HRPA at
pH 5.5 (Table 1) in sodium acetate buffer at 258C. As
Table 1: Binding of C-, J-, and M-containing PNA to RNA HRPA.[a]
PNA
Acetate
Acetate
Phosphate
pH 7.4[c]
PNA/RNA
stoichiometry
(Supporting
Information,
pH 5.5[b]
pH 7.0[b]
Table S1), which was consistent with triple-helix formation.
UV thermal melting experiments (Supporting Informa-
tion, Figure S15) confirmed the ITC results. Consistent with
our previous observations,[4] the complexes of HRPA and high
affinity PNAs melted in one transition of triple helix to single
strands without an intermediate duplex. In phosphate buffer
at pH 7.4 adding PNA2 had little effect on the stability of
HRPA: tm = 758C for HPRA alone and 748C for a 1:1
complex of HRPA-PNA2. Consistent with the higher Ka
observed in the ITC experiments, the thermal stability of
a 1:1 complex of HRPA-PNA3 was significantly higher at
808C. Taken together, the results confirmed our hypothesis
that the charged M would have an advantage over the neutral
J for triple-helical recognition of RNA.
PNA1 (C)
PNA2 (J)
PNA3 (M)
0.76
–
–
0.06
0.41
36.5
NB
0.17
1.8
[a] Association constants Ka ꢀ107 mÀ1, NB=no binding, Ka <103.
[b] 100 mm Sodium acetate buffer at 258C. [c] 2 mm MgCl2, 90 mm KCl,
10 mm NaCl, 50 mm potassium phosphate at 378C.
expected, because of the unfavorable protonation of cytosine
at higher pH, the affinity decreased significantly when the pH
of the buffer was increased to 7 and we could not observe any
binding in phosphate buffer mimicking the physiological
conditions at 378C.
We used the affinity of PNA1 at pH 5.5 as a benchmark to
gauge the effect of J and M modifications on PNA affinity at
higher pH. The affinity of J-modified PNA2 for HRPA in
acetate buffer at pH 7 was lower than the affinity of PNA1 at
pH 5.5 and decreased even more under the more demanding
physiological conditions (Table 1). Nielsen and co-workers[20]
Next we probed the sequence specificity of M-modified
PNA using a model system from our previous studies
(Figure 3).[4,5] Table 2 shows that PNA5 (four M modifica-
tions) had high affinity for the matched HRP1 at physiolog-
ically relevant conditions while maintaining excellent
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relevant double helical RNA. MicroRNAs (miRNAs) are
transcribed as long hairpin structures, pri-miRNAs, which are
processed into mature miRNA duplexes (ca. 22 nt) by Drosha
and Dicer endonucleases. It is common to find stretches of
eight and more contiguous purines interrupted by one or two
pyrimidines in pri-miRNA hairpins.[26] Triple-helical binding
to such sites could be used to detect miRNAs and interfere
with their function, which would find broad applications in
fundamental science, medicine, and biotechnology. We chose
HRP6 as a model that contains the purine-rich recognition
site present in pri-miRNA-215.[26] HRP6 has a stretch of nine
purines interrupted by a uridine and features several non-
canonical base pairs, which are hallmarks of pri-miRNA
hairpins. For recognition of the uridine interruption, we used
nucleobase E (Figure 4) that was originally designed for
thymidine recognition in DNA[8] and later adopted by us for
uridine recognition in RNA.[5] PNA7 having three M and one
E modification was prepared using monomers 2 and the
previously reported[5] 6 (Figure 4).
Figure 3. PNA, RNA, and DNA used in the sequence specificity study.
Table 2: Binding of M-modified PNA to RNA hairpins.[a]
PNA
HRP1
HRP2
HRP3
HRP4
(G-C)
(A-U)
(C-G)
(U-A)
PNA4[b]
PNA5[c]
PNA6[c]
8.4
2.0
0.04
<0.001[d]
0.4
0.05
NB[e]
0.02
NB[e]
NB[e]
NB[e]
NB[e]
[a] Association constants Ka ꢀ107 mÀ1. [b] From Ref. [4], in 100 mm
sodium acetate buffer, pH 5.5 at 258C. [c] In phosphate buffer, pH 7.4 at
378C. [d] Highest estimate; the low binding prevented more accurate
curve fit. [e] NB=no binding, Ka <103.
sequence specificity. The binding affinity of M-modified
PNA5 at pH 7.4 was somewhat lower than that of unmodified
PNA4 at pH 5.5.[4] This was in contrast to a similar compar-
ison of M-modified PNA3 and unmodified PNA1 in Table 1.
The discrepancy might be related to electrostatic repulsion of
adjacent charged nucleobases, which has been reported to
have a negative effect on affinity,[23] and may affect PNA5
more than PNA4. Nevertheless, the strong and highly
selective RNA binding by PNA5 at physiologically relevant
conditions was extremely encouraging. UV thermal melting
(Supporting Information, Figure S25) of the matched PNA5-
HRP1 complex showed a broad and relatively weak transition
at about 558C that might be assigned to triplex melting
preceding the melting of the HRP1 hairpin at about 1008C.
Similar transitions unique to the matched PNA5-HRP1
complex were also observed in CD melting plots (Supporting
Information, Figure S26). Consistent with the high sequence
selectivity, we could not observe any transitions above 308C
that could be assigned to triplex melting of the mismatched
complexes. Confirming the unique RNA selectivity observed
for PNA3, PNA5 showed little, if any binding to its matched
DNA hairpin HRP5 (Supporting Information, Figure S24).
PNA6 (three M modifications) had five times lower
affinity for the matched HRP2 than PNA5 for HRP1, which
was consistent with a higher stability of triplets involving G–C
base pairs and the notion that the positive charges are
important for high binding affinity. As expected, PNA6
showed excellent sequence specificity. The PNA-RNA stoi-
chiometry was 1:1 in all experiments shown in Table 2 (see
also the Supporting Information, Table S1).
Figure 4. Binding of E- and M-modified PNA7 to HRP6 to model the
pri-miRNA-215 hairpin structure.
Consistent with results obtained with other M-modified
PNAs, PNA7 recognized HRP6 with high affinity (Ka = 1.2 ꢀ
107) and 1:1 stoichiometry (Supporting Information,
Table S1) under physiologically relevant conditions. Remark-
ably, the non-canonical C*A and A*A and the wobble UoG
base pairs did not prevent formation of the PNA-RNA
complex.
In summary, we have demonstrated that modification of
PNA with 2-aminopyridine (M) nucleobases allows formation
of stable and sequence selective triple helices with double-
stranded RNA at physiologically relevant conditions. For
triple-helical RNA recognition, modulation of nucleobase
basicity (cf. pKa = 6.7 for M with 4.5 for C) was a more
efficient approach than using the neutral J base. The M-
modified PNAs exhibited unique RNA selectivity and had
two orders of magnitude higher affinity for the double
stranded RNAs than for the same DNA sequences. It is
conceivable that the deep and narrow major groove of RNA
presented a better steric fit for the PNA ligands than the
wider major groove of DNA. In preliminary experiments,
nucleobase-modified PNA recognized a purine-rich model
Finally, we chose microRNA-215, which is implicated in
cancer development and drug resistance,[24,25] as an initial
target to check if M-modified PNA could bind to biologically
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Published online: November 4, 2012
Keywords: modified nucleobases · peptide nucleic acids ·
.
sequence-selective RNA recognition · triple helices
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