Structural design and synthesis of bimodal PNA that simultaneously binds two complementary DNAs to Form fused double duplexes

Article abstract of DOI:10.1021/acs.orglett.0c01950

Bimodal PNAs are new PNA constructs designed to bind two different cDNA sequences synchronously to form double duplexes. They are synthesized on solid phase using sequential coupling and click reaction to introduce a second base in each monomer at Cα via alkyltriazole linker. The ternary bimodal PNA:DNA complexes show stability higher than that of individual duplexes. Bimodal PNAs are appropriate to create higher-order fused nucleic acid assemblies.

Full text of DOI:10.1021/acs.orglett.0c01950

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Letter
Structural Design and Synthesis of Bimodal PNA That
Simultaneously Binds Two Complementary DNAs To Form Fused
Double Duplexes
Manoj Kumar Gupta, Bharath Raj Madhanagopal, Dhrubajyoti Datta, and Krishna N. Ganesh*
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Supporting Information
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ABSTRACT: Bimodal PNAs are new PNA constructs designed to
bind two different cDNA sequences synchronously to form double
duplexes. They are synthesized on solid phase using sequential
coupling and click reaction to introduce a second base in each
monomer at Cα via alkyltriazole linker. The ternary bimodal
PNA:DNA complexes show stability higher than that of individual
duplexes. Bimodal PNAs are appropriate to create higher-order fused
nucleic acid assemblies.
eptide nucleic acids (PNA) are designed acyclic analogues
Pof DNA endowed with an ability to recognize and bind
complementary DNA/RNA sequences to form PNA:DNA and
PNA:RNA duplexes.1 The achiral PNA backbone with
repeating units of aminoethylglycine (aeg) has nucleobases
(A/T/C/G) connected via tertiary amide group (Figure 1a).2
The structural geometry of aeg backbone in PNA ensures
matching of the interbase distance in PNA with that of
adjacent bases on the sugar−phosphate backbone of DNA/
RNA,3 leading to sequence-specific complementary base
pairing between the PNA and the DNA/RNA complements
(Figure 1b). PNA:DNA duplexes have strong binding and
superior stability, compared to that of natural DNA:DNA
duplexes.4 This, along with unique strand invasion properties,5
has allowed PNA to be used for several biotechnological
applications in diagnostics6 and antisense therapeutics.7 The
simplicity of PNA construct provides abundant opportunities
for rational design of its structure to improve its properties8
and cellular uptake.9,10
Substitutions on aeg backbone at Cα, Cβ, and Cγ does not
seriously impair the hybridization with cDNA/cRNA and,
depending on the nature of substitution, PNAs acquire
additional functions.10,11 This manuscript describes the design
of iso-PNA (Figure 1c) carrying nucleobases (A/G/C/T) on
side chain at Cα linked through a triazole ring, instead of t-
amide link. The interbase distance in iso-PNA is similar to that
in original PNA and can be structurally fused with original
PNA (Figure 1a) to generate “bimodal PNA” composed of two
faces (Figure 1d). This equips a single PNA strand to
recognize and concurrently bind two different DNA strands to
produce double duplexes (Figure 1e) with a common PNA
backbone.
Synthetic “Janus bases” known in literature recognize two
natural bases, one from each face12 and PNAs composed from
such synthetic bases have innovative applications.13 The
Received: June 10, 2020
Figure 1. Structures of (a) aeg-PNA, (b) PNA:DNA duplex, (c) iso-
PNA, (d) bimodal PNA (bm-PNA), and (e) bimodal PNA double
duplex. B = T/A/G/C.
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bimodal PNA “bm-PNA” (Figure 1d) described here are
conceptually different with PNA backbone itself acquiring
Janus character. bm-PNA is designed to bind cDNA/RNA/
PNA as single strands or two strands simultaneously through
canonical base pairing. It is demonstrated that bm-PNA forms
double duplexes by simultaneous binding of two comple-
mentary DNA strands (Figure 1e) and the stability of the
DNA:bm-PNA:DNA ternary complex is enhanced over that of
isolated duplexes.
Scheme 1. Structures of Monomers 1−4 and Their
Synthesis
The target iso-PNA 1 and bm-PNA 2 (Figure 2) have
nucleobases attached to Cα(S) of each glycyl component on
Figure 2. Structures of iso-PNA 1 and bm-PNA 2.
aeg backbone through an ethyl triazole spacer (Figure 2). In
bm-PNA 2 this is in addition to the standard t-amide linked
nucleobases as in PNA. The triazole side chain at Cα design
was chosen more due to ease of synthesis than any structural or
functional considerations. iso-PNA 1 (Figure 2) bearing only
nucleobases anchored on Cα side chain through triazole was
used to test the ability of this new PNA analogue for base
pairing with cDNA. The monomers 1-4 possessing Cα (S)-
ethylazido side chain for subsequent click reaction were
synthesized from easily available L-glutamine through inter-
mediate steps via compounds 5−10 (Scheme 1). The
monomers 11 (B = CCbz/ACbz/GiBu) needed for assembling
iso-PNA 1 were synthesized from azide 12 and base propynes
14−16 by click chemistry. The bimodal PNA monomer 17
having 2 bases per aeg unit was made by coupling 1 with 15.
The aeg-PNA-C monomer 18 was made by standard
procedures.14
Scheme 2. Solid-Phase Synthesis of Bimodal PNA bm-PNA
2
a
bm-PNA 2 with mixed sequence of five bases on t-amide side
and four bases on triazole side was assembled by solid-phase
synthesis on MBHA resin (Scheme 2), using well established
protocols.14 The first coupling on resin was done with standard
PNA monomer 18 to avoid possible steric effects with
modified monomers. Each coupling of Cα-ethylazido PNA
monomers (1−3) on resin was followed by click reaction with
appropriate base propynes (14−16). At an intermediate step,
the preclicked bimodal PNA monomer 17 having T at the t-
amide side and A on the triazole side was used to test strategy
for future development of a general approach for synthesis of
any mixed bm-PNA oligomers. The iso-PNA 1 was similarly
a
Ⓐ Monomer coupling: (A/T/G/C; 3 equiv), dry DMF (500 μL)
HOBt (3 equiv), HBTU (3 equiv), and DIPEA (3 equiv); Ⓑ Click
reaction: resin (10 mg) in dry DMF:pyridine (1:1), alkyne (9 mg, 6
equiv), CuI (12 mg, 18 equiv), ascorbic acid (3 mg, 5 equiv), DIPEA
(12 μL); and Ⓒ Cleavage: resin 10 mg, thioanisole (20 μL), 1,2-
ethanedithiol (8 μL), TFA (200 μL), TFMSA (16 μL). Compound
numbers at each step are taken from Scheme 2. For details regarding
the reaction procedures, see the Supporting Information.
synthesized on solid phase using monomers 11 (B = CCbz
/
ACbz/GiBu). After the solid-phase synthesis, the resin bound iso-
PNA 1 and bm-PNA 2 product oligomers were deprotected
and cleaved from the resin, purified by reverse-phase HPLC
and characterized by MALDI-TOF spectra (see the Supporting
Information).
values extracted from the peak in the first derivative curves are
shown in Figure 5. The iso-PNA 1:DNA 1 duplex (Figure 3A)
showed Tm of 42.8 °C, just for a 5-mer duplex (Figure 5A).
Since it has cationic amino backbone, it is possible that it can
also bind anionic DNA due to electrostatic interactions. To
The successful formation of antiparallel PNA:DNA triazole
duplexes from iso-PNA 1 and bm-PNA 2 (Figures 3A−E) was
shown by sigmoidal transitions observed in a temperature−UV
absorbance plot14,15 (see Figure 4). The melting point (Tm)
B
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test the sequence specificity of iso-PNA 1 binding to DNA 1,
its complementation was done with DNA 1m (see legend in
Figure 5) to yield duplex iso-PNA 1:DNA 1m, which has a C-T
mismatch and exhibited a significantly lower Tm (24.7 °C).
This suggests that iso-PNA 1:DNA 2 duplex arises from
specific base pairing and not mere electrostatic interactions.
The bm-PNA 2 formed corresponding triazole duplex bm-
PNA 2:DNA 1 (Figure 3B) with Tm of 48.7 °C (Figure 5B)
that is slightly higher than that of iso-PNA 1:DNA 1 triazole
duplex. bm-PNA 2 also gave t-amide face duplex DNA 2:bm-
PNA 2 (Figure 3C) with Tm = 30.2 °C (Figure 5C), which is
lower than that of its triazole duplex. Thus, the designed bm-
PNA 2 forms complementary base-paired duplexes independ-
ently from both triazole and t-amide faces. The triazole face
duplexes A and B (Figure 3) result from new isomorphic
bimodal PNA structure, in which the nucleobases are attached
at Cα, quite different from that of standard PNA.
The designed bimodal PNA bm-PNA 2, in the presence of
DNA 1 and DNA 2 yielded the ternary complex DNA 2:bm-
PNA 2:DNA 1 (Figure 3D) made from two duplexes. This
complex showed a single melting transition (Figure 4b) with
Tm of 56.6 °C, which is significantly higher than the
constituent isolated duplexes DNA 2:bm-PNA 2 and bm-
PNA 2:DNA 1 (Figure 5). The double duplex does not show a
sequential melting (as in DNA triplexes16), but displays
simultaneous dissociation of both DNA strands from bm-PNA
2 similar to PNA2:DNA triplex.2,17 The bimodal PNA
backbone being common to both duplexes couples the
unwinding of one duplex to influence that of other duplex,
causing melting of both duplexes to be synchronous and not
sequential. The coexistence of duplexes with common
backbone in a single complex strengthens their mutual stability,
impacting dissociation of both duplexes. These results provide
proof of concept that as per the design, bimodal PNA
concomitantly binds to two DNA strands that are comple-
mentary to triazole and t-amide face.
Figure 3. iso-PNA 1 and bm-PNA 2 duplexes: (A) iso-PNA 1: DNA
1; (B) bm-PNA 2:DNA 1; (C) DNA 2:bm-PNA 2; (D) DNA 2:bm-
PNA 1:DNA 2 open double duplex and (E) DNA 3hp:bm-PNA 2
hairpin double duplexes.
Creation of double duplex from bm-PNA 2, DNA 1 and
DNA 2 (Figure 3D) is a termolecular reaction requiring
entropic cost. The two complementary DNA segments were
connected by a tetranucleotide TTTT yielding single DNA
3hp. This can now bind both sides to yield hairpin double
duplex DNA 3hp:bm-PNA 2 (Figure 3E) as a binary complex.
This hairpin double duplex interestingly displayed two well-
resolved transitions with Tm values of 34.6 and 65.4 °C (Figure
4c), in contrast to open double duplex that showed a single
transition. The two transitions in hairpin duplex 3E arise either
from sequential melting of t-amide and triazole duplex, or the
early transition from a premelting conformational change,
followed by simultaneous disassociation of both strands.
Notably, both observed transitions in hairpin duplex 3E are
stabilized, compared to isolated duplexes 3B and 3C and that
of ternary complex 3D. Note that in isolated duplexes and
open double duplex 3D, both duplexes are in antiparallel
orientation. In hairpin double duplexes, the triazole duplex
segment is in parallel orientation and hence stabilities are not
directly comparable. The PNA:DNA transitions are generally
known to be broad due to slow association−disassociation
kinetics,14 and in bimodal PNA duplex and double duplexes,
they are even more broad.
Figure 4. UV-melting plots and CD of duplexes. (a) A, iso-PNA
1:DNA 1; B bm-PNA 2:DNA 1; and C DNA 2:bm-PNA 2. (b) D,
DNA 2:bm-PNA 2: DNA 1 and (c) E DNA 3hp:bm-PNA 2 (red line
represents normalized melting curves and black dashed line represents
first derivative plots). (d) CD profiles of duplexes D and E.
Figure 5. Tms for various bimodal PNA:DNA duplexes and double
duplexes, A−E correspond to complexes as in Figure 3. Values
accurate to 1.0 °C. iso-PNA 1:DNA 1m Tm = 24.7 °C, Mismatch
DNA sequence: DNA 1m = 5′-CGCGGA-3′.
The CD spectra of open double duplex D and hairpin
double duplex E are shown in Figure 4d. The profiles resemble
that observed for PNA:DNA duplexes,18 indicating similar
overall conformational features. Both D and E show a positive
C
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a
Table 1. Thermodynamic Parameters from ITC
duplex
bm-PNA:DNA
−ΔG (kcal/mol)
−ΔH (kcal/mol)
−ΔS (cal/(mol K))
KD (× 10−6 M)
B
C
D
E
bm-PNA 2:DNA 1 (triazole)
DNA 2:bm-PNA 2 (t-amide)
DNA 2:bm-PNA 2:DNA 1
DNA 3hp:bm-PNA 2
6.67
6.52
6.22
7.49
33.7
26.1
37.9
24.8
94.0
68.0
109.7
60.0
8.78
11.4
19.5
2.11
a
For experimental details, see Figures S10−S12 in the Supporting Information.
CD band at ∼280 nm, with a slight difference in negative band
position: 260 nm for the open duplex D and 250 nm for
hairpin duplex E. The CD band intensities were also higher for
hairpin duplex E, compared to open duplex D, suggesting a
better stacking in the hairpin duplex. The temperature-
dependent CD of hairpin double duplex E (Figure S9 in the
Supporting Information) showed slight shifts in maximum at
280 nm, accompanied by decreased intensity with an isosbestic
point at ∼265 nm, indicating alteration in conformation during
melting. The stacking of triazole rings concomitant with base
pairing may provide additional stability in bimodal PNA
duplexes. The results establish formation of double duplexes
from bm-PNA 2 and biophysical mechanisms of melting of
bimodal PNAs need further study.
The thermodynamic parameters for formation of duplexes
and double duplexes in bimodal PNAs were sought from ITC
(see Table 1, as well as Figures S10−S12 in the Supporting
Information). The experiment with bimodal PNA double
duplex D was performed by titrating an equimolar mixture (in
terms of the number of bases/binding sites) of DNA 1 and
DNA 2 into bm-PNA 2. The dissociation constants (KD)
indicated the binding of triazole duplex B to be slightly
stronger than t-amide duplex C, but association is entropically
less favored (more negative). The stacking of triazole rings
concomitant with canonical base pairing contributes to higher
enthalpy, providing additional stability in the formation of bm-
PNA 2 duplex B.
Formation of termolecular double duplex D, is accompanied
by an entropic cost (more negative entropy), but offset by
having a higher enthalpic contribution. Notably, the hairpin
duplex E is most favored in terms of lowest KD and lowest
(−ΔS) and highest (−ΔG) among different complexes. The
negative ΔG for bimodal PNA:DNA duplexes in the range of
−6.0 kcal/mol to −7.0 kcal/mol was similar to that reported
for PNA:DNA duplexes.19
In summary, this manuscript presents new concept of
bimodal PNA that can concurrently hybridize with two
cDNAs. In the isomorphic PNA backbone iso-PNA 1,
nucleobases anchoring on Cα(S) glycyl side chain successfully
form triazole DNA duplex. The base pairing in this new
isomorphic PNA duplex is likely to be Watson−Crick (WC)
type, since it shows sequence specificity with lower Tm in
mismatched duplex. The bimodal bm-PNA 2 designed to
simultaneously bind two complementary DNAs forms open
and hairpin double duplexes from t-amide and triazole faces.
These coexist in a ternary complex with stability higher than
the individual binary duplexes. The double duplex dissociates
in a single step similar to PNA2:DNA triplexes,17 while the
hairpin double duplex shows distinct premelting conforma-
tional change before disassociation.16 This arises from the fact
that a single common backbone hosts two duplexes. Based on
choice of sequences (polypurines/polypyrimidines/self-com-
plementary) on either side, bimodal PNAs can lead to fused
duplexes, triplexes, and tetraplexes (Gn/Cn) with cDNA. They
can be engineered to yield supramolecular functional
nanostructures for probable applications in PNA material
science.20 Distant biotechnological applications include simul-
taneous targeting of two genes or micro RNA structures21 for
therapeutic purposes, and, as equivalents of short DNA staples,
they can be active in programmed folding of plasmids in DNA
origami.22
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01950.
Synthesis procedures, characterization data (NMR, MS
of all new compounds, HPLC, MALDI TOF of PNA
oligomers), UV-melting curves, CD spectra, ITC data
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
Krishna N. Ganesh − Department of Chemistry, Indian Institute
of Science Education and Research (IISER) Pune, Pune
411008, India; Department of Chemistry, Indian Institute of
Science Education and Research (IISER) Tirupati, Tirupati
517507, India; orcid.org/0000-0003-2292-643X;
Email: kn.ganesh@iisertirupati.ac.in
Authors
Manoj Kumar Gupta − Department of Chemistry, Indian
Institute of Science Education and Research (IISER) Pune, Pune
411008, India
Bharath Raj Madhanagopal − Department of Chemistry,
Indian Institute of Science Education and Research (IISER)
Tirupati, Tirupati 517507, India; orcid.org/0000-0003-
1043-8754
Dhrubajyoti Datta − Department of Chemistry, Indian Institute
of Science Education and Research (IISER) Pune, Pune
411008, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01950
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.N.G. acknowledges the Department of Science and
Technology (DST), New Delhi for a research grant (No.
EMR/2016/007601). M.K.G. thanks UGC, New Delhi for a
research fellowship. B.M. thanks IISER Tirupati, and D.D.
thanks DST (Delhi) for postdoctoral fellowships.
D
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a structurally biased backbone. Updated review and emerging
challenges. Curr. Top. Med. Chem. 2011, 11, 1535−1554. (f) Moccia,
M.; Adamo, M. F. A.; Saviano, M. Insights on chiral, backbone
modified peptide nucleic acids: Properties and biological activity.
Artif. DNA PNA XNA 2014, 5, e1107176.
REFERENCES
■
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Sequence
selective recognition of DNA by strand displacement with thymine-
substituted polyamide. Science 1991, 254, 1497−1500.
(2) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. Peptide
Nucleic Acids (PNA). Oligonucleotide analogues with an achiral
peptide backbone. J. Am. Chem. Soc. 1992, 114, 1895−1897.
(3) Egholm, M.; Nielsen, P. E.; Buchardt, O.; Berg, R. H. J.
Recognition of guanine and adenine in DNA by cytosine and thymine
containing peptide nucleic acids (PNA). J. Am. Chem. Soc. 1992, 114,
9677−9678.
(9) (a) Koppelhus, U.; Nielsen, P. E. Cellular delivery of peptide
nucleic acid (PNA). Adv. Drug Delivery Rev. 2003, 55, 267−280.
́
(b) Rownicki, M.; Wojciechowska, M.; Wierzba, A. J.; Czarnecki, J.;
Bartosik, D.; Gryko, D.; Trylska, J. Vitamin B12 as a carrier of peptide
nucleic acid (PNA) into bacterial cells. Sci. Rep. 2017, 7, 7644.
(c) Gasparello, J.; Manicardi, A.; Casnati, A.; Corradini, R.; Gambari,
R.; Finotti, A.; Sansone, F. Efficient cell penetration and delivery of
peptide nucleic acids by an argininocalix[4]arene. Sci. Rep. 2019, 9,
3036−3045.
(4) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier,
S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
PNA hybridizes to complementary oligonucleotides obeying the
Watson−Crick hydrogen-bonding rules. Nature 1993, 365, 566−568.
(5) (a) Nielsen, P. E.; Christensen, L. Strand displacement binding
of a duplex forming homopurine PNA to a homopyrimidine duplex
DNA target. J. Am. Chem. Soc. 1996, 118, 2287−2288. (b) Demidov,
V. V.; Yavnilovich, M. V.; Belotserkovskii, B. P.; Frank-Kamenetskii,
M. D.; Nielsen, P. E. Kinetics and mechanism of polyamide ‘peptide’
nucleic acid binding to duplex DNA. Proc. Natl. Acad. Sci. U. S. A.
1995, 92, 2637−2641.
(6) (a) Ray, A.; Norden, B. Peptide nucleic acid (PNA): its medical
and biotechnical applications and promise for the future. FASEB J.
2000, 14, 1041−1060. (b) Nielsen, P. E. PNA technology. Mol.
Biotechnol. 2004, 26, 233−248. (c) Lundin, K. E.; Good, L.;
Stromberg, R.; Graslund, A.; Smith, C. I. E. Biological activity and
biotechnological aspects of peptide nucleic acid. Adv. Genet. 2006, 56,
1−51. (d) D’Agata, R.; Giuffrida, M. C.; Spoto, G. Peptide nucleic
acid-based biosensors for cancer diagnosis. Molecules 2017, 22, 1951−
1966. (e) Vilaivan, T. Fluorogenic PNA probes. Beilstein J. Org. Chem.
2018, 14, 253−281. (f) Saarbach, J.; Sabale, P. M.; Winssinger, N.
Peptide nucleic acid (PNA) and its applications in chemical biology,
diagnostics and therapeutics. Curr. Opin. Chem. Biol. 2019, 52, 112−
124. (g) Economos, N. G.; Oyaghire, S.; Quijano, E.; Ricciardi, A. S.;
Saltzman, W. M.; Glazer, P. M. Peptide nucleic acids and gene
editing: Perspectives on structure and repair. Molecules 2020, 25,
735−736.
(7) (a) Good, L.; Nielsen, P. E. Progress in developing PNA as a
gene-targeted drug. Antisense Nucleic Acid Drug Dev. 1997, 7, 431−
437. (b) Doyle, D. F.; Braasch, D. A.; Janowski, B. A.; Corey, D. R.
Inhibition of gene expression inside cells by peptide nucleic acids:
Effect of mRNA target sequence, mismatched bases, and PNA length.
Biochemistry 2001, 40, 53−64. (c) Quijano, E.; Bahal, B.; Ricciardi, A.;
Saltzman, W. M.; Glazer, P. M. Therapeutic peptide nucleic acids:
Principles, limitations, and opportunities. Yale J. Biol. Med. 2017, 90,
583−598. (d) Montazersaheb, S.; Hejazi, M. S.; Charoudeh, H. N.
Potential of peptide nucleic acids in future therapeutic applications.
Adv. Pharm. Bull. 2018, 8, 551−563. (e) Lee, H. T.; Kim, S. K.; Yoon,
J. W. Antisense peptide nucleic acids as a potential anti-infective
agent. J. Microbiol. 2019, 57, 423−430. (f) Gambari, R.; Gasparello, J.;
Finotti, A. Peptide nucleic acid-based targeting of microRNAs:
possible therapeutic applications for glioblastoma. J. Cancer Metastas.
Treat. 2019, 5, 55−62. (g) Smith, C. I. E.; Zain, R. Therapeutic
oligonucleotides: State of the art. Annu. Rev. Pharmacol. Toxicol. 2019,
59, 605−630.
(8) (a) Hyrup, B.; Egholm, M.; Nielsen, P. E.; Wittung, P.; Norden,
B.; Buchardt, O. Structure-activity studies of the binding of modified
peptide nucleic acids (PNAs) to DNA. J. Am. Chem. Soc. 1994, 116,
7964−7970. (b) Kumar, V. A.; Ganesh, K. N. Conformationally
constrained PNA analogues: Structural evolution toward DNA/RNA
binding selectivity. Acc. Chem. Res. 2005, 38, 404−412. (c) Govindar-
aju, T.; Kumar, V. A.; Ganesh, K. N. (SR/RS)-Cyclohexanyl PNAs:
Conformationally preorganized PNA analogues with unprecedented
preference for duplex formation with RNA. J. Am. Chem. Soc. 2005,
127, 4144−4155. (d) Kumar, V. A.; Ganesh, K. N. Structure-editing
of nucleic acids for selective targeting of RNA. Curr. Top. Med. Chem.
2007, 7, 715−726. (e) Corradini, R.; Sforza, S.; Tedeschi, T.;
Totsingan, F.; Manicardi, A.; Marchelli, R. Peptide nucleic acids with
(10) (a) Mitra, R.; Ganesh, K. N. PNAs grafted with (α/γ, R/S)-
aminomethylene pendants: Regio and stereospecific effects on DNA
binding and improved cell uptake. Chem. Commun. 2011, 47, 1198−
1200. (b) Mitra, R.; Ganesh, K. N. Aminomethylene peptide nucleic
acid (am-PNA): synthesis, regio-/ stereospecific DNA binding, and
differential cell uptake of (α/γ, R/S) am-PNA analogues. J. Org. Chem.
2012, 77, 5696−5704. (c) Jain, D. R.; Anandi V, L.; Lahiri, M.;
Ganesh, K. N. Influence of pendant chiral Cγ-(alkylideneamino/
guanidino) cationic side-chains of PNA backbone on hybridization
with complementary DNA/RNA and cell permeability. J. Org. Chem.
2014, 79, 9567−9577. (d) Ellipilli, S.; Palvai, S.; Ganesh, K. N.
Fluorinated peptide nucleic acids with fluoroacetyl side chain bearing
5-(F/CF3)-uracil: Synthesis and cell uptake studies. J. Org. Chem.
2016, 81, 6364−6373. (e) Ellipilli, S.; Vasudeva Murthy, R.; Ganesh,
K. N. Perfluoroalkylchain conjugation as a new tactic for enhancing
cell permeability of peptide nucleic acids (PNAs) via reducing the
nanoparticle size. Chem. Commun. 2016, 52, 521−524.
(11) (a) Dragulescu-Andrasi, A.; Rapireddy, S.; Frezza, B. M.;
Gayathri, C.; Gil, R. R.; Ly, D. H. A simple gamma-backbone
modification preorganizes peptide nucleic acid into a helical structure.
J. Am. Chem. Soc. 2006, 128, 10258−10267. (b) Ishizuka, T.; Yoshida,
J.; Yamamoto, Y.; Sumaoka, J.; Tedeschi, T.; Corradini, R.; Sforza, S.;
Komiyama, M. Chiral introduction of positive charges to PNA for
double-duplex invasion to versatile sequences. Nucleic Acids Res. 2008,
36, 1464−1471.
(12) (a) Branda, N.; Kurz, G.; Lehn, J.-M. Janus wedges: A new
approach towards nucleobase-pair recognition. Chem. Commun. 1996,
2443−2444. (b) Zhao, H.; Huang, W.; Wu, X.; Qing, Y.; Xing, Z.; He,
Y. Synthesis of a complete Janus-type guanosine-cytosine base and its
2’-deoxyribonucleoside. Chem. Lett. 2011, 40, 684−686. (c) Chen, H.;
Meena; McLaughlin, L. M. A Janus-wedge DNA triplex with A-W1-T
and G-W2-C base triplets. J. Am. Chem. Soc. 2008, 130, 13190−
13191. (d) Shin, D.; Tor, Y. Bifacial nucleoside as a surrogate for both
T and A in duplex DNA. J. Am. Chem. Soc. 2011, 133, 6926−6929.
(e) Artigas, G.; Marchan, V. Synthesis of Janus compounds for the
recognition of G-U mismatched nucleobase pairs. J. Org. Chem. 2013,
78, 10666−10677.
(13) (a) Xia, X.; Piao, X.; Bong, D. Bifacial peptide nucleic acid as an
allosteric switch for aptamer and ribozyme function. J. Am. Chem. Soc.
2014, 136, 7265−7268. (b) Thadke, S. A.; Hridya, V. M.; Perera, J. D.
R.; Gil, R. R.; Mukherjee, A.; Ly, D. H. Shape selective bifacial
recognition of double helical DNA. Comm. Chem. 2018, 1, 79.
(c) Thadke, S. A.; et al. Design of bivalent nucleic acid ligands for
recognition of RNA-repeated expansion associated with Huntington’s
disease. Biochemistry 2018, 57, 2094−2108.
(14) (a) Bentin, T.; Hansen, G. I.; Nielsen, P. E. Measurement of
PNA binding to double-stranded DNA. In Methods in Molecular
Biology; Nielsen, P. E., Ed.; Humana Press: Totowa, NJ, 2002; Vol.
208, pp 91−109. (b) Nielsen, P. E., Ed. Peptide Nucleic Acids Protocols
and Applications, 2nd Edition; Horizon Bioscience: Norfolk, U.K.,
2004.
(15) (a) Sen, A.; Nielsen, P. E. Unique properties of purine/
pyrimidine asymmetric PNA-DNA duplexes: Differential stabilization
of PNA-DNA duplexes by purines in the PNA strand. Biophys. J.
́
2006, 90, 1329−1337. (b) Jasinski, M.; Miszkiewicz, J.; Feig, M.;
E
https://dx.doi.org/10.1021/acs.orglett.0c01950
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Trylska, J. Thermal stability of peptide nucleic acid complexes. J. Phys.
Chem. B 2019, 123, 8168−8177.
(16) Plum, D. E.; Park, Y.-W.; Singleton, S. F.; Dervan, P. B.;
Breslauer, K. J. Thermodynamic characterization of the stability and
the melting behavior of a DNA triplex: A spectroscopic and
calorimetric study. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 9436−9440.
(17) Krupnik, O. V.; Guscho, Y. A.; Sluchanko, K. A.; Nielsen, P. E.;
Lazurkin, Y. S. Thermodynamics of the melting of PNA2/DNA triple
helices. J. Biomol. Struct. Dyn. 2001, 19, 535−542.
(18) Corradini, R.; Tedeschi, T.; Sforza, S.; Marchelli, R. Electronic
Circular Dichroism of Peptide Nucleic Acids and Their Analogues. In
Comprehensive Chiroptical Spectroscopy, Vol. 2: Applications in
Stereochemical Analysis of Synthetic Compounds, Natural Products, and
Biomolecules; Berova, N., Polavarapu, P. L., Nakanishi, K., Woody, R.
W., Eds.; John Wiley & Sons, Inc, 2012; pp 581−617.
(19) Ratilainen, T.; Holmen, A.; Tuite, E.; Nielsen, P. E.; Norden, B.
Thermodynamics of sequence-specific binding of PNA to DNA.
Biochemistry 2000, 39, 7781−7791.
(20) (a) Manicardi, A.; Rozzi, A.; Korom, S.; Corradini, R. Building
on the peptide nucleic acid (PNA) scaffold: a biomolecular
engineering approach. Supramol. Chem. 2017, 29, 784−795.
(b) Duan, T.; He, L.; Tokura, Y.; Liu, X.; Wu, Y.; Shi, Z.
Construction of tunable peptide nucleic acid junctions. Chem.
Commun. 2018, 54, 2846−2850. (c) Swenson, C. S.; Velusamy, A.;
Argueta-Gonzalez, H. S.; Heemstra, J. Bilingual peptide nucleic Acids:
Encoding the languages of nucleic acids and proteins in a single self-
assembling biopolymer. J. Am. Chem. Soc. 2019, 141, 19038−19047.
(d) Canady, T. D.; Berlyoung, A. S.; Martinez, J. A.; Emanuelson, C.;
Telmer, C. A.; Bruchez, M. P.; Armitage, B. A. Enhanced
hybridization selectivity using structured gamma PNA probes.
Molecules 2020, 25, 970−982.
(21) Cadoni, E.; Manicardi, A.; Madder, A. PNA-based microRNA
detection methodologies. Molecules 2020, 25, 1296−1321.
(22) (a) Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami:
Scaffolds for creating higher order structures. Chem. Rev. 2017, 117,
12584−12640. (b) Bila, H.; Kurisinkal, E. E.; Bastings, M. M. C.
Engineering a stable future for DNA-origami as a biomaterial.
Biomater. Sci. 2019, 7, 532−541.
F
https://dx.doi.org/10.1021/acs.orglett.0c01950
Org. Lett. XXXX, XXX, XXX−XXX