Custom PNA Oligos

Due to high affinity and specificity, PNA has superior hybridization efficiency and sensitivity.

PNA Applications

• Sequence specific PCR blocker (PNA clamp)
• FISH probes for telomere, centromere, gene specific probes, microorganisms
• Anti-sense/ anti-microbial reagents
• miRNA inhibitors
• Double strand DNA invasion & capture
• Microarray probes
• Quantification of RNA therapeutics

• Antisense and antigene therapy

PNA can bind with high specificity to complementary RNA or DNA sequences, blocking translation or transcription. This makes it a promising candidate for gene silencing or correction in therapeutic applications.

• siRNA quantification

PNA probes can be used in hybridization-based assays (e.g., LC-FD) for highly specific detection and quantification of siRNA in biological samples.

• PCR clamping and blocking

PNAs can suppress amplification of unwanted sequences (e.g., wild-type alleles) during PCR, allowing selective amplification of rare or mutant alleles with high sensitivity.

• FISH (Fluorescence In Situ Hybridization)

PNA probes are used in FISH for detecting specific DNA sequences in chromosomes with high signal-to-noise ratio and rapid hybridization, making them ideal for telomere or centromere analysis.

• PNA–peptide conjugates

Linking PNA to cell-penetrating peptides (CPPs) facilitates intracellular delivery for gene targeting or antimicrobial applications. Conjugates can also include nuclear localization signals or other functional moieties.

• Double-stranded DNA invasion & capture

PNA can invade double-stranded DNA by forming stable PNA–DNA–PNA triplexes or duplexes, displacing one DNA strand. This allows highly specific binding without prior denaturation and is useful for gene targeting, mutation detection, or isolating genomic regions for downstream analysis.

• PNA molecular beacons

These are PNA probes labeled with a fluorophore and quencher that fluoresce upon hybridization, enabling real-time detection of specific nucleic acid targets in live cells or diagnostics.

• Molecular diagnostics

Due to its strong binding and mismatch discrimination, PNA is widely used in detecting single nucleotide polymorphisms (SNPs), mutations, or pathogen sequences in clinical diagnostics.

• Biosensors and nanotechnology

PNA’s stability and specificity make it an excellent component for biosensors, often immobilized on surfaces or nanoparticles to detect target nucleic acids in complex samples.

 

General Guidelines for PNA Oligomers

1. Design the PNA oligomer to be complementary to the intended DNA or RNA target, just like standard DNA primers. For double-stranded DNA targets, either strand can be used. When possible, evaluate both orientations and select the sequence with the most favorable binding and solubility characteristics.
2. PNA oligomers bind to complementary DNA or RNA targets with significantly higher affinity than standard oligonucleotides. The T_m of PNA is about 1°C per base pair higher than DNA. There is about a 15°C T_m difference for a single-base mismatch for PNA, while it is about 10°C for DNA. PNA binds most strongly to a complementary PNA target, followed by RNA and then DNA.
3. PNA with a high purine content (>50%), a high amount of G bases (>35%), a long purine stretch (>7 bp), or a longer length (>30 bp) may exhibit reduced solubility in aqueous solutions. For applications limited to aqueous solution, keep these constraints in mind. For applications utilizing organic solvents, such as FISH, these constraints do not apply.

Solubility Tip

Consider adding solubility enhancers such as two lysine residues or O linkers. For double-stranded DNA targets, reverse-complement sequences can be used to avoid high purine content.

4. Due to the neutral backbone of PNA, the binding of PNA to nucleic acid is less dependent on ionic strength and pH. PNA binding to DNA or RNA is not affected significantly from 10 mM to 1 M NaCl concentration.
5. Please refer to the PNA Tool on our website for design guidelines and T_m calculation.

 

Handling and Storage of PNA Oligomers Upon Receipt

1. Briefly centrifuge the tube before opening.
2. Dissolve PNA to prepare a 20–100 µM stock. For example, dissolving 50 nmol of PNA in 500 µL of solvent results in a 100 µM stock. Water is generally the recommended solvent for PNA. For FISH, formamide is a better alternative. Avoid Tris buffer, as PNA tends to have lower solubility.
3. To help ensure complete dissolution, heat the PNA solution at 55°C for 5 minutes and mix thoroughly. If the PNA is not fully dissolved, heat the PNA at 85°C for an additional 5 minutes. Lowering the PNA concentration may also improve solubility. At typical working concentrations of 1 µM or lower, most PNAs should remain soluble in aqueous assay solutions.
4. If the PNA is still not fully dissolved, consider the following options:
   • For applications that can include organic solvents, add DMSO or DMF to a final concentration of 10–20%, or up to 50%. Repeat the heating step if necessary.
   • For applications that cannot use organic solvent, add glacial acetic acid to a final concentration of 0.1–1% v/v.
   • For the most difficult PNA to dissolve, 0.1% trifluoroacetic acid, TFA, v/v and 20% DMSO may be used.
5. Store in aliquots at 4°C for short-term use, for up to one month. Storing PNA at −20°C or −70°C is recommended for long-term storage. Protect fluorescently labeled probes from light.
6. After taking the PNA solution out of cold storage, heat it at 55°C for 5 minutes and mix thoroughly before use.
7. PNA is highly stable and should remain intact for more than three years when properly stored.

Recommended Stock Preparation

• Recommended stock concentration: 20–100 µM
• Recommended solvent: water
• For FISH applications: formamide may be used
• Avoid Tris buffer when possible
• Typical working concentration: 1 µM or lower

 

PNA Quantification

Calculate the molar extinction coefficient, ε₂₆₀, of the PNA oligomer. If the PNA is conjugated to a fluorophore or another chemical that contributes to absorbance at 260 nm, include its contribution in the calculation.

Extinction Coefficient Formula

ε₂₆₀ (PNA oligomer) = Σ ε_i × n_i

• i = A, G, C, and T
• ε_i = molar extinction coefficient of the base
• n_i = number of each base

Base	ε260 [mL/(µmol × cm)]
A	13.7
C	6.6
G	11.7
T	8.8

 

1. Calculate the molar extinction coefficient, ε₂₆₀, of the PNA oligomer. Include the contribution of a fluorophore or other chemical modification if it contributes to absorbance at 260 nm.
2. If needed, transfer a known volume of the PNA stock solution and dilute it to an appropriate final volume. Calculate the dilution factor.
3. Measure the absorbance of the PNA solution at 260 nm using a spectrophotometer.
4. Calculate the PNA concentration using the formula below.

PNA Concentration Formula

PNA concentration (mM) = [A₂₆₀ ÷ (ε₂₆₀ × path length in cm)] × dilution factor

Due to its high specificity and affinity, PNA oligomers effectively bind to their target nucleic acids. Unlabeled PNAs can be used as gene-specific blockers in PCR reactions (PNA clamping). PNA clamping can efficiently detect SNP mutations in a target gene as PNA can distinguish a single base mismatch.

When designing PNA oligomers for use in aqueous solution-based applications such as PCR, the following guidelines are recommended to ensure good solubility:

• Purine content <50%
• G bases <35%
• PNA length <23 bp
• Purine stretches <6 residues

For double-strand DNA targets, a reverse complement sequence can be chosen for PNA design. If there is no flexibility in PNA design, solubility enhancers such as two O linkers or two Lysines can be incorporated into the PNA.

Please refer to PNA TOOL for Tm predictions and other guidelines.

To learn more about PNA clamping technology and the PNA clamp products available as catalog items, please visit PCR blockers.

Our technical team would be happy to review your PNA sequences or help with the PNA design. Quotation requests, inquiries, and orders can be directed to order@pnabio.com.

PNA oligomers can be labeled at the 5′ and/or 3′ ends. It is recommended to include 1-2 O linkers (also known as eg1 or AEEA linkers) as a spacer between the label and the PNA. Since 3′ end PNA (-CONH2) cannot be directly modified, one Lysine is added and its primary amine is used for conjugation for the 3′ labeling.

A wide range of labels are available, including fluorophores such as FAM, FITC, Alexa dyes, Atto dyes, Cy dyes,HEX, ROX, Thiazole Orange (-NHS ester or carboxy amide), as well as quenchers like BHQ and Dabcyl, and other functionalities such as Acridine, Alkyne (DBCO or Pentynoic acid), Azide, Biotin, Digoxigenin, Ferrocene, Maleimide, Methylene Blue, Myristol, Palmitic acid, Pacific Blue, Thiol, and more.

A PNA probe with a fluorophore at the 5′ end and a quencher at the 3′ end can function as a molecular beacon (MB). Unlike DNA, the flexible structure of PNA allows it to remain in a quenched state without requiring a stem-loop design in unbound form, making probe design shorter and simpler. A wide range of fluorophore and quencher pairs are available. Thanks to the high specificity and binding affinity of PNA, molecular beacons offer highly sensitive detection without the need to wash away unbound probes.

For custom conjugation, please send an email to order@pnabio.com.

 

Dye	Ex (nm)	Em (nm)	Dye	Ex (nm)	Em (nm)
FAM	492	518	Atto425	436	484
FITC	492	518	Alexa Fluor 488	494	517
Oregon Green 488	498	526	TAMRA	553	576
Cy3	550	570	Texas Red	596	615
Alexa Fluor 546	556	573	Atto550	554	574
Alexa Fluor 532	530	555	Atto647N	644	667
Cy5	650	670	Alexa Fluor 647	650	668
Thiazol Orange	510	530	Dabcyl	454
JOE	520	548	BHQ-2	560-670
BHQ-1	480-580		BHQ-3	620-730

Because the PNA backbone is polyamide-based, it can be readily conjugated to peptides for added functionality. For instance, adding lysine residues can improve solubility, while cysteine can enable conjugation via disulfide bond formation.  

Peptides may be attached to either the 5′ or 3′ end of the PNA, with an O-linker optionally included as a spacer between the peptide and the PNA strand. 

 PNA–peptide conjugates have a range of applications. In antimicrobial research, PNA sequences (typically 10–15 bases) targeting essential microbial genes can be paired with cell-penetrating peptides (CPPs) like (KFF)3K or (RXR)4XB for intracellular delivery. In mammalian cells, antisense PNAs are often designed to target mRNA regions near the 5′ ATG start codon. In addition, nuclease localization signl (NLS) can be conjugated to facsilitate transport of PNA into nucleus. Bipartite PNA-peptide can also efficiently capture target microorganisms.  

 We can synthesize PNA-peptide conjugates up to 50 residues in length via standard linear synthesis. For longer constructs, PNA and peptide can be synthesized separately and conjugated by maleimide-cysteine ligation.  

For inquiries or to place an order, please contact us at order@pnabio.com. 

Gamma PNA contains an R group at a stereogenic center at the γ-carbon of the N-aminoethyl glycine unit. The addition of gamma modifications increases the melting temperatures (Tm) by 5-8 °C , enhancing sequence-specific binding and enabling invasion into double-stranded DNA. Gamma PNA also provides benefits such as improved solubility, reduced self-aggregation, stable duplex formation with DNA, and flexibility for multi-labeling and other functionalization.

• Possible gamma functional groups
  – Lysine: better solubility, possible for dual-labeling, the potential for cell penetration
  – Alanine and glutamic acid are also possible modifications.
  – Lysine: enhance solubility, possible for dual-labeling, the potential for cell penetration
  – Glutamic acid: adds negative charges, improves solubility
  – Alanine: increases hydrophobicity, can be added to every residue
  – Serine and miniPEG: improves solubility without altering charges

Sensitive siRNA quantification

1. A novel hybridization LC‑MS/MS methodology for quantification of siRNA in plasma, CSF and tissue samples. Yuan L et al. (2023). Molecules 28(4) 1618.
2. Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis. Hutanu A et al. (2023) Analytical Chemistry 95 (11): 4914-4922.
3. Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles. Biscans A et al. (2021) Molecular Therapy 29(4):1382–1394. 
4. Quantitative determination of a siRNA (AD00370) in rat plasma using peptide nucleic acid probe and HPLC with fluorescence detection. Tian Q et al. (2017) Bioanalysis 9(11):861–872

PNA peptide therapeutics

1. Endosomolytic Peptides Enable the Cellular Delivery of Peptide Nucleic Acids. Giancola JB & Raines RT (2024) bioRxiv In press.
2. Antisense inhibition of RNA polymerase α subunit of Clostridioides difficile . Pal R & Seleem MN (2023) Microbiol Spectr 11(5):e0175523.
3. Antimicrobial activity of antisense peptide–peptide nucleic acid conjugates against non-typeable Haemophilus influenzae in planktonic and biofilm forms. Otsuka T & Murphy TF (2017) Microbiol Spectr. 72: 137. Review.
4. Dual Repression of the Multidrug Efflux Pump CmeABC by CosR and CmeR in Campylobacter jejuni . Grinnage-Pulley T et al. (2016) Front Microbiol 7:1097.

PNA clamping

1. Peptide Nucleic Acid-Mediated Regulation of CRISPR-Cas9 Specificity. Carufe KEW et al. (2024) Nucleic Acid Ther In press.
2. An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine. Zheng Z et al. (2023)Sensors & diagnostics 2(1): 163.
3. Bacterial Communities Show Algal Host (Fucus spp.) Zone Differentiation Across the Stress Gradient of the Intertidal Zone. Quigley CTC et al (2020) Front Microbiol 11: 563118.
4. Plasmonic Nanoparticle Conjugation for Nucleic Acid Biosensing. Tadimety A et al. (2022) Methods Mol Biol 2393: 73-87.
5. Design of peptide nucleic acid probes on plasmonic gold nanorods for detection of circulating tumor DNA point mutations. Tadimety A et al. (2019) Biosensors & bioelectronics Vol.130, p.236-244.

PNA FISH

1. Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by superresolution microscopy. Tommaso ED et al. (2023) Mol Biol Cell 34(6): ar61.
2. Quantification and Localization of Protein-RNA Interactions in Patient-Derived Archival Tumor Tissue. Blanchard EL et al. (2023) Cancer research (Chicago, Ill.) 79(20): 5418-5431.
3. Combating Antimicrobial Resistance via Single-Cell Diagnostic Technologies Powered by Droplet Microfluidics. Hsieh K et al. (2022) Accounts of Chemical Research 55(2):123.
4. Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens. Mach KE et al. (2019) Analyst 144(5): 1565.
5. Fluorescence spectroscopic detection and measurement of single telomere molecules. Beh CW et al. (2018) Nucleic Acids Res 46(19): e117.
6. Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay. Godinho BM et al. (2017) Nucleic Acid Therapeutics 27 (6): 323.

ctDNA detection

1. Engineering of a DNA/ ΓPNA Hybrid Nanoreporter for ctDNA Mutation Detection via ΓPNA Urinalysis. Xiang Z et al. (2024) Adv Sci e2310225.
2. Development of a Detection System for ESR1 Mutations in Circulating Tumour DNA Using PNA-LNA-Mediated PCR Clamping. Kojima Y et al. (2023) Diagnostics (Basel) 13(12):2040, 2023.
3. Dual-recognition-based determination of ctDNA via the clamping function of peptide nucleic acid and terminal protection of small-molecule-linked DNA. Chen C et al. (2020) Analyst 145(23):7603-7608, 2020.

The cost of custom oligos varies based on length, quantity, modifications, and other requirements. Synthesis typically takes 2-3 weeks for standard cases and an additional week for gamma PNA or special labeling. Minimum order quantities are 50 nmole for unlabeled PNA and 25 nmole for labeled PNA. Feel free to check out PNA Tool for Tm predictions and other guidelines for PNA design.

For quotes and orders, inquiries can be directed to order@pnabio.com.