Custom PNA Oligos

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

PNA Application

• 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.

 

For most applications, we recommend PNA probes that are 15–18 bases in length, with a melting temperature (Tm) around 75°C and a purine content below 60%.

For aqueous-based applications such as PCR, it is best to use sequences with purine content under 50%, G content below 35%, and total length under 23 bases. For applications involving organic solvents such as FISH or FD-LC, purine content should be 75% or lower. If your target is double-stranded DNA, consider using the reverse complement sequence.

Linkers are commonly used as a spacer when conjugating PNA with other functional groups like peptides or dyes. Hydrophilic linkers such as O, E, and X linkers can enhance the solubility of PNA oligomers. Several types of linkers are possible. The O linker (also known as AEEA or eg1) is most commonly used spacer, which also improve solubility. Other spacers such as E, X, C3, C4, C6, and C12 linkers are also available. C6SH or C11SH can be used for adding a thiol group to the PNA.

If you have no flexibility in PNA sequence, adding two lysines or O-linkers can improve solubility.

Possible linkers

• O linker (also called AEEA or eg1): improve solubility, most commonly used
• As a spacer: O, E, X, L35, C4, C6, C12 linker
• Thiol addition: C6SH, C11SH

O-linker (9 atoms, 1.3 nm)

E-linker

C6A linker (7 atoms, 1.1 nm)

C6SH

X-linker

C11SH (12 atoms, 1.9 nm)

 

To calculate Tm and check other properties, please use PNA Tool.

If you are selecting a PNA oligomer from a target region longer than 20 bases, PNA Designer Tool can help narrow down optimal sequences.

Our team is happy to review your chosen sequences or assist with custom design. Feel free to contact us at order@pnabio.com.

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.