Custom PNA Oligos

Due to its high affinity and specificity, PNA oligos can efficiently bind its target nucleic acid. One of the popular usages of unlabeled PNA is a gene specific blocker of PCR reaction (PNA clamp). This technology can efficiently detect SNP mutations in a target gene.

PNA binds to its target nucleic acid by either orientation but antiparallel is preferred over parallel. As a default, PNA is written from 5' to 3' or N- to -C, just like DNA or peptides. It is also written from NH2- to -CONH2 or H to -NH2. The 5' end of PNA NH2- can be conjugated to other functional groups while the 3' end of PNA -CONH2 is an inactive end. Acetylation at 5' end can block any potential reactivity.

Because Tm of PNA is higher than that of DNA, usually 10~21mer is used for most applications. A longer length of PNA can reduce solubility. High purine content (>60%), especially G base can be a reason for low solubility. Depending on the sequence, the possible longest length of PNA is about 40mer. However, it is strongly recommended to check its Tm and design the probe that has appropriate length and sequence.

Please refer PNA TOOL for Tm and other guidelines.

To improve the solubility of PNA probes, the addition of solubility enhancers such as O linker, E linker, X linker, or 2 Lysines is recommended for PNA with long length (>22mer) or high purine content (>60%). These linkers can also work as a spacer for conjugation to other functional groups such as peptides or dyes.

Possible linkers

    - O linker (also called AEEA or eg1): improve solubility, most commonly used
    - As a spacer: O, E, X, C3, 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)

PNA oligomers can be labeled at 5' and/or 3' end. Since 3' end PNA is inactive (-CONH2), one Lysine is added and its amine is used for conjugation at 3' labeling.

It is recommended to include 1~2 O linkers (also called eg1, or AEEA linker) between the label and PNA for 5' end labeling.

Possible labels: most fluorophores such as FAM, FITC, Alexa dyes, Atto dyes, Cy dyes, ROX, Thiazole Orange (-NHS ester or carboxy amide), quenchers (BHQ, Dabcyl), Acridine, Alkyne (DBCO or Pentynoic acid), Azide, Biotin, Digioxygenin, Dabcyl, Ferrocene, Maleimide, Methylene Blue, Myristol, Palmitic acid, Pacific Blue, Thiol, etc

For custom conjugation, please contact us.

Since the backbone of PNA is based on poly amides, PNA can be easily linked to peptide to add functionality. For example, Lysine addition can improve the solubility of PNA. The addition of Cysteine can be used as a way to conjugate other molecules using disulfide bond formation.

A peptide can be conjugated at 5' end or 3' end but 5' end conjugation (peptide-PNA) is more popular. O linker can be added between peptide and PNA as a spacer.

One of the most popular applications of PNA peptide conjugation includes anti microbicidal reagents, which comprise of CPP (most commonly (KFF)3K or (RXR)4XB) and 10~15 bp of PNA molecules that are antisense to the essential gene of the microbes.

Similarly in mammalian cells, an anti-sense oligo approach can be easily adapted using CPP and PNA conjugation where PNA is designed antisense to its target mRNA. Most commonly, PNA is designed to 5' ATG and upstream region.

Another approach using peptide and PNA conjugation is in microarray type where you can capture both antigene and transcript from the target microorganisms.

In general, PNA peptide conjugates are consecutively synthesized on resin from C-terminus to N-terminus.

Gamma PNA has a stereogenic center at the γ-carbon atom of the N-aminoethyl glycine unit. γ-substituted PNA can be placed in every third residue of regular PNA.

Tm of gamma PNA is higher by 5~8 ℃ per single substitution, which results in more sequence-specific binding at higher affinity. In addition, gamma PNA can invade double-stranded DNA to form a triple helix.

Moreover, gamma PNA can provide several advantages such as improved solubility, less self-aggregation, more stable PNA-DNA duplex formation, and flexibility for multi labeling and other functionalization.

    - Lysine: better solubility, possible for dual-labeling, the potential for cell penetration
    - Alanine and glutamic acid are also possible modifications.