1

Peptide Nucleic Acid (PNA)
23/10/26 Literature Seminar
Mayu Onoda

Contents

What is PNA?

2

What is PNA?

3

PNA (= Peptide Nucleic Acid)…「Peptide」 + 「Base」
BaseA
PeptideGCT

2-aminoethyl glycine

DNA mimic

Peter E. Nielsen et al. Science, 1991, 254, 1497

Le, Bao T. et al. Molecular Therapy - Nucleic Acids, 2018, 14, 142

What is PNA?

4Invasion

・Probe・Gene Expression Regulation
TargetPNA✔ Electrically neutral✔ Resistant to nuclease / protease✘ Low cell membrane permeability
Jacques, S et al. Current Opinion in Chemical Biology, 2019, 52,112

Contents

5

PNA application in Chemical Biology
Fluoresce labeling by using PNA

Protein

PNATag

In cellUlrike Reinhardt. et al. Bioconjugate Chemistry, 2015, 26, 10
Gavins, G.C., et al. Nat. Chem., 2021, 13, 15
Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

PNA application

6☆Labeling
TargetProteinProtein
Coil＋PNA
Coil

PNA

Already reported“Peptide Coil-Coil Interaction”
Gradišar, H. and Jerala, R, 2011, J. Peptide Sci., 17, 100

PNA

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

ProteinPNA application

7Fluorescence

DNAWithin 2minProtein
Protein

PNA

Native Chemical Ligation

PNA<PNA – Coil structure>

Coil
Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

PNA application
Fluoresce labeling
Target Protein

8

EGFR
Gavins, G.C., Nat. Chem., 2021, 13, 15.
Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

EGFR (Epidermal Growth Factor Receptor)

EGFR

・Cell membrane protein
・Ligand-activated (EGF ligand)・Related to signaling pathways,
cell proliferation, differentiation and survival

Maron Steven B., et al. Frontiers in Oncology, 2020, 10

PNA application

9【Protocol】
EGFR

EGFR expressionin HEK293 cell

Coil

HEK293cell

GFP
Tag

Coil-Coil(100 nM,25℃, 4min)

NativeChemical
Ligation

Atto565 / Cy3(200 nM, 25℃, 5min)

PNA-EGFR
Fluorescent labeling
Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

EGFR
GFP

PNA

PNA application

10Atto565or Cy3✔Specific labelingon cell membrane

No PNA (Negative control)✔No labelinga, Schematic structures of the Cys-E3-EGFR-eGFP construct and the PNA-tagging reagents PNA11K3 and PNA16-K3 used for labelling. b–f, After staining of nuclei with Hoechst 33342 (shown in blue),transiently transfected cells were treated with PNA11-K3 (b,f) or PNA16-K3 (d,g). For control, PNA tagging
was omitted in c and e. Subsequently, cells were incubated with TMR-labelled PNA-12mer (b,c), Cy3labelled DNA-16mer (d,e), Cy3-labelled DNA-12mer (f) or Atto565-labelled DNA-16mer (g) single strands.
Conditions for PNA transfer: (1) 0.1 mM TCEP in PBS (pH 7.0), 2 min, 25 °C; (2) 100 nM donor PNAnK3 in PBS buffer (pH 7.0), 4 min, 25 °C. Conditions for hybridization: 200 nM TMR-/Cy3-/Atto565-labelled
PNA/DNA strands in PBS (pH 7.0), 5 min, 25 °C. Scale bars, 10 μm. All experiments were repeated threetimes independently with similar results.

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

PNA application

11[Coil1-EGFR][Coil2-ErbB2]
expression in CHO cell
PNA1 / PNA2

Coil-Coil(100 nM, 4min)

NativeChemicalLigation

PNA1-EGFR
PNA2-ErbB2
Fluorescent labeling

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

(200 nM, 4min)

PNA application

12HPLC analysis(B) matched and (C) mismatched coiled coil peptides. Conditions: 200 nM Cys-P1-TMR or Cys-P3-TMR, 1200nM PNA1-P2 or PNA3-P4 in 200 nM phosphate, 1 mM TCEP, 0.1% CHAPS, pH 7.2, 30 °C. (D) One-pot
reactions of a Cys-P1-TMR/Cys-P3-C343/PNA1-P2/PNA3-P4-mixture (red traces) overlaid with Fl-UPLC tracesfor reactions involving Cys-P1-TMR + PNA1-P2 or Cys-P3-C343 + PNA3-P4 in separate pots (black traces).
Detector settings: TMR, Ex 550 nm, Em 580 nm; C343, Ex 420 nm, Em 500 nm.

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

✔ Mismatch reactions
do not occur.

PNA application

13

Atto565

EGFR

PNA1

YFP

Cy7

ErbB2

PNA2

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

Dual color live cell labeling. After nuclear staining with Hoechst33342 (blue), (A) Cys-P1-EGFR-eYFP or (B) Cys-P3-ErbB2-eCFP
expressing CHO cells were treated with matched (left) or mismatched (right) PNA labeling reagents/fluorophore-DNA. Conditions: 4min with 100 nM PNA1-P2 (EGFR) or/and PNA3-P4 (ErbB2) in DPBS, washing with HBSS, 4 min hybridization with 200 nM
Atto565-DNA1 (EGFR) or Cy7-DNA3 (ErbB2). (C) Simultaneous labeling of Cys-P1-EGFR-eYFP/Cys-P3-EGFR-eCFP cells. Scalebar = 10 μm.

CFPPNA application

14①F②ProteinF①Protein
Already reported.

PNA

What’s the merit?

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

Ulrike Reinhardt. et al. Bioconjugate Chemistry, 2015, 26, 10

PNA application

15<PNA merit ①>
Increasing of fluorescence intensity
DNA

Targetprotein

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

DNA(50 nM)

PNA

PNA

Cy7(100 nM)

Cy7

CHO cellTag10533
GCG TCA TCC AGA GTC CTA CTG GTA AGT GGT GTC

GCG TCA TCC AGA GTC CTA CTG GTA AGT GGT GTCcgc agt agg tct cag

GAC CAT TCA CCA CAGcgc agt agg tct cag

151515GAC CAT TCA CCA CAG

155

PNA application
Cy7165 × Cy7✔ Fluorescence intensity
increasea, Labelling of CHO cells stably expressing Cys-P1-EGFR-eYFP by PNA tagging and subsequenthybridization with a single Cy7-DNA (Complex I, adaptor DNA 33mer with one Cy7-15mer, left) or five
Cy7-DNA strands (Complex II, adaptor DNA 105mer with five Cy7-15mers, right). Red arrows indicate
the lines on which line intensity profiles in Fig. 4b are measured. PNA transfer: 100 nM donor PNA15P2 in HBSS buffer, 4 min, 25 °C. Hybridization: 50 nM DNA complex in HBSS-BB, 4 min, 25 °C. The
brightness of images depicting 1× Cy7 labelling was digitally increased relative to 5× Cy7 for clarity.
For images showing 1× Cy7 and 5× Cy7 labelling with equal brightness settings, see
Supplementary Fig. 9-6. Scale bars, 20 μm. Experiments were repeated three times independently
with similar results.

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

c, Dependence of staining intensity on the number of Atto647N dyes in complexes used for staining (DNA105mer + 1, 3 or 5 equiv. of Atto647N-DNA-15mer; Complexes IV, V and VI, respectively). After labelling as
described in a, cells were detached, fixed and analysed by flow cytometry to determine the ratio of mean
Atto647N and YFP fluorescence intensity values, after gating was applied (Supplementary Fig. 12-1). Data arepresented as the mean ± SD of n = 3 independent experiments.

PNA application

17<PNA merit ②>
Fluorescence Elimination / Replacement
PNA15Elimination
DNA15+8DNA 50 nM, 4min

Atto56515+8300 nM
15+8 2×5min

DNAGavins, G.C., et al. Nat. Chem., 2021, 13, 15.

PNA application

18<PNA merit ②>
Fluorescence elimination / replacement

Replacement
Cy715
100 nM
3min

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

PNA application

19✔Elimination✔Replacement

a–c, Wide-field images of reversible labelling without EGF stimulation. a, After staining nuclei with Hoechst 33342 in HBSS-BB,
serum-starved Cys-P1-EGFR-eYFP cells were treated with PNA15-P2 in HBSS for 4 min. Cells were incubated with 50 nM
Complex III (adaptor DNA-105mer with five Atto565-DNA-23mers containing a toehold for strand displacement) in HBSS-BB for
4 min. Subsequently, 50 nM Complex II (adaptor DNA-105mer carrying five Cy7 DNA15mer strands) and 50 nM Cy7 DNA-15mer
were added for 4 min. b, Toehold-mediated strand displacement of the ATTO565-23mer DNA was carried out with 300 nM
displacement DNA-23mer for 2 × 5 min in HBSS at 30 °C. The brightness of the Atto565 images in rows a and b was digitally
increased to give ‘overexposed’ images, which illustrate that Atto565 labelling was completely removed in b. c, 100 nM Cy7
DNA-15mer was hybridized for 3 min to illustrate restaining.

Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

PNA application

20⑤③ DNA erasers

Replacement①Ligand
EGF
EGFR

EGFR
Elimination④② internalize

Label only internalized proteins
Separate labeling possible
Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

PNA application

21(100 nM)

Internalization
stimulusIN

Elimination

OUT

Replacement

Figure 9-7 Widefield fluorescence microscope characterization of PNA-tag enabled reversible
fluorescence labelling of Cys-P1-EGFR-eYFP on serum starved CHO cells, stimulated with EGF. After
staining of nuclei with Hoechst 33342 in HBSS-BB, cells were treated with PNA15-P2 in HBSS, 4 min. A)
Cells were incubated with 50 nM Complex III; adaptor DNA-105mer with five Atto565-DNA-23mers; in
HBSS-BB for 4 min. B) 100 nM EGF stimulation for 15 mins C) Toehold mediated strand displacement ofAtto565-23mer DNA with 300 nM displacement DNA-23mer and 100 nM EGF for 2 x 5 min in HBSS at 30°C.
D) 100 nM Cy7-DNA-15mer, 3 min. E) The same experiment was carried out without addition of PNA orDNA.BB: Blocking Buffer: 0.1mg/mL salmon sperm DNA, 0.2% BSA, 1x ProLong™ Live Antifade in HBSS.
Excitation times: Cy7: 500 ms, ATTO565: 50 ms YFP: 50 ms, Hoechst 33342:10 ms. Filter settings: Hoechst
33342) λex = 350 ± 50 nm, λem = 460 ± 50 nm; YFP) λex = 500 ± 24 nm, λem 545 ± 40 nm, Cy7) λex = 710 ±75 nm, λem 810 ± 90 nm. Scale bar= 20 μm. Experiments were repeated 3 times independently with similar
results.

✔Separate intracellularand extracellular detection
Gavins, G.C., et al. Nat. Chem., 2021, 13, 15.

Short summary

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Fluorescent labelingｃ・・・

PNAProtein
Tag

In cell「Peptide」 + 「Base」✔ Separate labeling of two proteins
fluorescence✔ Increasing✔ Elimination✔ Replacement

Short summary

23

Target：Cell membrane protein

The Low cell membrane permeability
PNA
is not much of a problem.

In cell PNA application(Example: Gene expression regulation)

Improvement of cell membrane permeability
is needed.

Contents

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Improvement ofPNA cell membrane permeability△PNAIn cell

Improvement of PNA cell membrane permeability

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CPP (=cell-penetrating peptide) strategy
BaseBaseBaseー ー+Guanidine
Side chain+(1 μM GPNA, 37℃, 5min 10×Guanidine PNA)

GPNA
DAPI

Peng, Z. et al. J. Am. Chem. Soc., 2003, 125, 6878
Anca, D. A, et al. J. Am. Chem. Soc. 2006, 128, 16104

FITC

Overlay

Figure S4. Fluorescent images of HCT 116 cells following: (i) incubation with 1µM GPNA for 5min at 37oC, (ii) thoroughly washed with PBS, (iii) fixed with 4%paraformadehyde for 30min at room temperature, (iv) permeabilized with 1%
Triton-X for 30min, (v) incubate with 1uM DAPI for 30min, (vi) thoroughly
washed and mounted on the microscope slide. A: image taken with DAPIchannel, B: image taken with FITC channel (emission from fluoresceincovalently linked to GPNA), and C: an overlay between A and B.

Improvement of low cell membrane permeability

26

Gene Expression Regulation (E-cadherin)…… GTGGCTGCAGCCAGGT(Complementary E-cadherintranscription start site)

E-cadherin localization in A549 cells
GPNA (-) , 72 h

GPNA 10 μM, 72 h

Decreased protein levels.
Figure 2 E-Cadherin immunofluorescent-stainingof the untreated (B) and treated cells (C−H).
A549 cells were treated with 10 μM of GPNA for
72 h and then stained with E-cadherin primary
antibody and counterstained with FITC-labeledsecondary antibody. The identity of eacholigomer is indicated in the parentheses.

Anca, D. A, et al. J. Am. Chem. Soc. 2006, 128, 16104

Improvement of low cell membrane permeability

Guanidine

VS27

Poly Arg
xR

Poly Arg
8R
EthD-1 (red, dead cells)
calcein (green, live cells)

4R

Guanidine

6R 8R 10R
(10 μM, 24 h)( x μM, 48 h)✔ Guanidine side chain ⇒ Low toxicity
Figure 7 Cytotoxic effects of PNA conjugates and GPNAs. (A) The sequence of PNA conjugates and GPNAs. The configuration of arginine residues in PNA conjugates is D; bold letters denote GPNA units. (B) The effects ofoligomer size on cytotoxicity. Nearly confluent (80−90%) A549 cells were treated with 10 μM of the indicated PNA conjugates and GPNAs for 24 h and then stained with EthD-1 (dead cells) and calcein (live cells). (C) Exponentiallygrowing A549 cells were treated with various concentrations of 16-mer PNA conjugate (P16-8R) and GPNA (P16) for 48 h and then stained with EthD-1 and calcein. Representative fluorescent images of cells treated with 15 μMof P16-8R (D) and P16 (E) for 48 h and stained with EthD-1 (red, dead cells) and calcein (green, live cells).

Anca, D. A, et al. J. Am. Chem. Soc. 2006, 128, 16104

Improvement of low cell membrane permeability28+
Cationic motifs on base(1μM, 30min)

PNA

High cell membrane permeability
Park Soree, et al. Frontiers in Microbiology, 2023, 14(C) Cell permeability of FAM-OPNA in HCT-8 cells. HCT-8 cells were seeded in an 8-well
cell culture slide and incubated overnight. FAM-OPNA was treated at a concentration of
1 μM for 30 min, and nuclei were stained with DAPI for 20 min. Images were obtained by aconfocal laser microscope.

Tightly & selectively binds to pre-mRNA.
➡Regulate splicing
https://www.olipass.com/front/eng/competitive/technology.doSummary29

Improvement ofcell membrane permeability

In cellTarget

Appendix

30

PNA synthesis

31

PNA monomer

Base
Protecting group

SPPS

Skeleton synthesis of PNA monomer

A. Y. Shaikh. et al. Eur. J. Org. Chem. 2021, 2792.

Synthesis of Fmoc-protected PNA backbone intermediate 2:(a) 4 °C → room temp., 12 h, neat (56 %);(b) PTSA, toluene, reflux with Dean-Stark setup, 3 h (99 %);(c) MeOH−toluene (1 : 1), reflux at 110 °C, 12 h (94 %);(d) Fmoc−OSu, DIPEA, THF, 0 °C → room temp., 3 h (96 %).

PNA synthesis32

T

Fmoc-TGACT-NH2

C

Synthesis of Fmoc/Boc-protected PNA monomers(a) 2, TSTU, DMF, room temp., 12 h (to give 7 a, 91 %; 7 b, 87 %; 7 c, 83 %; or 7 d, 75 %);(b) LiOH, and then Fmoc−OSu, THF−H2O, pH 8, room temp., 3 h (to give 8 a, 90 %; 8 b, 92 %; 8 c, 90 %; or 8 d, 90 %).

33

AG

34

35

Coil-Coil36

疎水性電荷（＋/-）
https://onlinelibrary.wiley.com/doi/10.1002/psc.1331

37

Atto565

Excitation 545～575 nm
Cy7

DNA comlex

38

DNA

Atto56515+8DNA complexes were hybridized by premixing to a final
complex concentration of 10 μM in Hank’s balanced salt
solution (HBSS), heating to 70 °C and cooling to room
temperature over 30 min.

39

PNA application

EGFR

Ligand-activatedmembrane protein

40

ErbB2

Non-ligand-activatedmembrane protein

Internalization affects
cell signaling.

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

Frontiers | Targeting EGFR in Esophagogastric Cancer (frontiersin.org)

PNA application
GA (Geldanamycin)：
Hsp90 inhibitor
Destabilize protein

EGFR
Ligand-activatedmembrane protein

ErbB2
Non-ligand-activatedmembrane protein

Georgina, C. G, et al. RSC Chem. Biol., 2021, 2, 1291

41

Ligand stimulation
Promotes internalization

42

EGF刺激
Elimination

内在化と膜残留の判別ができない
膜上に残ったものが取り除かれたPNA11mer, TMRでの検出

43

Improvement of PNA cell membrane permeability

44

Tm (℃) = Melting temperature
PNA-DNA
PNA

GPNA-DNAHigh Tm→StableFigure S1. Melting curves of GPNA-DNA duplex and PNA2-DNA triplex. The
strand concentrations of DNA, PNA, and GPNA were 2µM, 4µM, and 2µM,
respectively. Buffer contained 10mM NaPi (pH 7.0) and 20mM NaCl. The
samples were first pre-annealed by incubating at 90Û&IRU PLQIROORZHGE¥ gradual-cooling to room temperature. UV-absorption wasmonitored at 260nm from 20 to 90Û&DW WKH rate of 0.5Û&SHUPLQXWH+ GPNA

Novel Binding and Efficient Cellular Uptake of Guanidine-Based Peptide Nucleic Acids (GPNA) | Journal of the American Chemical Society (acs.org

45

E-Cadherin

46

Cell adhesion moleculePNA structure

47

ProteinPNA15

Add cationic motif

48<Strategy> Guanine+ ( = G +)<Hypothesis> Ideal double-stand is formed?

Cationic guanine: positively charged nucleobase with improved DNA affinity inhibits self-duplex formation - Chemical Communications (RSC Publishing)Add cationic motif❏ Electrophoretic mobility shift assay✔ Increased invasion efficiency.
✔ Mismatch sequences are not recognized.

49Add cationic motif

50

Cationic guanine: positively charged nucleobase with improved DNA affinity inhibits self-duplex formation - Chemical Communications (RSC Publishing)How PNA works?

51❏dsDNA❏ssDNA / mRNAPNA

PNA’

PNA

Application・Gene editing
・DNA labeling

Application
・Regulation oftranscription and translation