Custom PNA Oligos
Custom PNA oligos can be unlabeled or labeled by dyes or other chemicals. They can also be conjugated to peptides to add other functionality. Linkers can be added as a spacer and also to improve solubility. Gamma PNA or modified bases can increase Tm and specificity of PNA even further.
PNA (Peptide Nucleic Acid) is an artificially synthesized polymer similar to DNA or RNA. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds as in peptides. Since PNA contains no charged phosphate groups, the binding between PNA and DNA is stronger than that between DNA and DNA due to the lack of electrostatic repulsion. PNA is resistant to DNases and proteases, and is extremely stable in vivo as well as in vitro.
PNA oligomers can form duplexes in either orientation, but the anti-parallel orientation is strongly preferred.
PNA/DNA-duplex generally has higher Tm than corresponding DNA/DNA-duplex. Roughly, there is increase of about 1oC per base pair.
Due to high binding affinity to its complementary DNA sequences, designs of long PNA oligomers are usually not needed for hybridization. PNA ologomers of 12-21 bases are most common.
lt is strongly recommended to avoid any self-complementary sequences such as inverse repeats, hairpin forming and palindromic sequences because PNA/PNA interaction is stronger than PNA/DNA interaction.
lt is also recommended to avoid purine-rich sequences because they tend to be aggregated as a result of low solubility in aqueous solution. Try to limit the purine content lower than 60%. Also try to avoid purine stretch over 6 residues, specially consecutive 4 or more G residues.
To improve solubility of PNA probes, addition of solubility enhancer such as O linker, E linker, X linker, or two Lysines can be used.
Please refer PNA TOOL for Tm and other guidelines.
Due to its high affinity and specificity, PNA oligos can efficiently bind its target nucleic acid. One of the popular usage 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. 5' end of PNA is NH2- (also written as 5' or H-) that can be conjugated to other functional groups, and 3' end of PNA is -CONH2 (also written as -NH2 or 3'), which is 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. Longer length of PNA can reduce solubility. High purine content (>60%), specially G base can be a reason for low solubility. Depending on the sequence, possible longest length of PNA is about 40 mer. 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 solubility of PNA probes, addition of solubility enhancer 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.
- As a spacer: O, E, X, C3, C4, C6, C12 linker
- Thiol addition: C6SH, C11SH
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, thiazole orange (-NHS ester or carboxy amide), biotin, alkyne (DBCO or pentynoic acid), palmitic acid, digioxygenin, dabcyl, BHQ, azide, methylene blue, thiol, maleimide, etc
For custom conjugation, please contact us.
|Dye||Ex (nm)||Em (nm)||Dye||Ex (nm)||Em (nm)|
|FITC||492||518||Alexa Fluor 488||494||517|
|Oregon Green 488||498||526||TAMRA||553||576|
|Alexa Fluor 546||556||573||Atto550||554||574|
|Alexa Fluor 532||530||555||Atto647N||644||667|
|Cy5||650||670||Alexa Fluor 647||650||668|
Since the backbone of PNA is based on poly amides, PNA can be easily linked to peptide to add functionality. For example, Lys addition can improve solubility of PNA. Addition of Cysteine can be used as a way to conjugate other molecules using disulfide bond formation.
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 antimicrocidal 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, 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-aminoethylglycine unit. γ-substituted PNA can be placed in every third residue of regular PNA.
Tm of gammaPNA is higher by 5~8 ℃ per single substitution, which result 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 duplexe formation, and flexibility for multi labeling and other functionalization.
- Lysine: better solubility, possible for dual labeling, potential for cell penetration
- MiniPEG: best for improved solubility and specific binding, efficient for double strand DNA invasion
- Alanine and glutamic acid are also possible modification.
Custom PNA oligos provided by PNA Bio will be provided >95% purity accompanied by COA including HPLC and mass analysis data.
The price of custom oligo is dependent on the length, amount and label. Please indicate the specifics in quote request. Feel free to check out PNA Tool for Tm calculation and other guidelines for PNA design. Synthesis usually takes 2~3 weeks for the most cases, and an extra week for gamma PNA or special labeling.
Minimum amount is 50 nmole for non-labeled PNA and 25 nmole for labeled PNA.