Custom PNA Synthesis
PNA (Peptide Nucleic Acid), an artificially created DNA analogue, was first invented by Professor Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark in 1991.
PNA has a structure in which the phosphate-ribose backbone of DNA is substituted with a peptide-like amide backbone (N-(2-aminoethyl) glycine). So the binding affinity and stability to the target DNA or RNA are greatly increased. Despite the structural change of the backbone, PNA can be used in a variety of applications where DNA can be used because it can make a complementary binding to the target sequence as DNA does.
PNA Applications
- Sequence-specific PCR blocker (PNA clamp)
- FISH probes for telomere, centromere, gene-specific probes, infection test
- Anti-sense/ anti-microbial reagents
- miRNA inhibitors
- Double strand DNA invasion & capture
- Microarray probes
Unlabeled PNA
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. 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 the inactive end. Acetylation at the 5' end can block any potential reactivity.
Because the Tm of PNA is higher than that of DNA, usually 10~21mer is used for most applications. The 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 40 mer. However, it is strongly recommended to check its Tm and design the probe that has the appropriate length and sequence.
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.
Labeled PNA
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 modifications:
- Fluorophore/Quencher: Cyanine dye; FAM; FITC; TAMRA (TMR); TexasRed; ROX; HEX; ATTO dye; Thiazol Orange; Methylene Blue; Dabcyl; BHQ; Digoxigenine; Biotin
- Linker: C3-NH2, C4-NH2; C6-NH2; C12-NH2; Polyethylene Glycol linker; E linker
- Functional group: Amine; Alkyne; Thiol; Maleimi; Azide; Acetylation
- Backbone modification: Alpha modification; Gamma modification
- Base modification: D (2,6-diaminopurine); J (Pseudoisocytosine); I (Inosine)
PNA-peptide
Since the backbone of PNA is based on polyamides, PNA can be easily linked to peptides to add functionality. For example, Lys 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.
The 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 basepair of PNA molecules that are antisense to the essential gene of the microbes.
Similarly in mammalian cells, the 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
Gamma (γ)-PNA is a backbone-modified PNA that possesses a stereogenic center through modifications introduced at gamma (γ)-carbon of the backbone. Due to the stereogenic center, the gamma-PNA oligo itself forms an alpha-helical structure, thereby reducing the self-aggregation, improving the solubility, and forming a more stable duplex with the target DNA. These features provide a higher binding affinity to the target. In addition, various modifications such as internal multi-labeling are possible at any gamma (γ)-position. With these advantages, gamma (γ)-PNA can be an attractive material for diagnostics and drug development.
Possible gamma functional groups
- Lysine: better solubility, possible for dual-labeling, the 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 modifications.
PNA Design
Binding properties: PNAs can form duplexes in either orientation, but the anti-parallel orientation is strongly preferred. This will be the orientation for all antisense and DNA probe type applications. The N-terminal of the PNA oligomer is equivalent to the 5'-end of an oligonucleotide and is often referred to as "the 5'-end of the PNA". A PNA/DNA-duplex will usually have a higher Tm than the corresponding DNA/DNA-duplex. As a rough rule, there will be an increase in Tm of about 1°C per base pair at 100 mM NaCl depending on the sequence.
Probe Length: Due to this higher affinity it is not necessary to prepare long PNA oligomers. For most applications an oligomer length of 12-18 is optimal. as opposed to the 25-40-mers, which is the typical length for an oligonucleotide probe. Bear in mind that the shorter a probe the more specific it is. The impact of a mismatch is greater, the shorter the sequence is. In many cases even shorter probes will work well, Longer PNA oligomers, depending on the sequence, tend to aggregate and are difficult to purify and characterize.
Purine Content: Purine-rich PNA oligomers tend to aggregate, with G-rich oligomers being the worst. As a rule, never have more than 7 purines in any stretch of 10 units. Observing this rule will dramatically reduce the likelihood that the PNA oligomer will aggregate. The shorter the sequence the less attention needs to be paid to the sequence design.
Self-complementarity: Avoid self-complementary sequences such as inverse repeats, hairpin forming, and palindromic sequences as PNA/PNA interactions are even stronger than PNA/DNA interactions. For example, AATT would be OK but not CCGG or ATTATT. There is no problem with the synthesis but they are generally difficult to characterize and purify.
PNA Order
- The price of custom PNA is dependent on the length, amount, and label. Please indicate the specifics in the quote request.
- Minimum amount is 20 nmole for non-labeled PNA and 10 nmole for labeled PNA.
- Custom PNA oligos will be provided at >90% or >95% purity by HPLC analysis along with the MALDI-TOF report.
- Synthesis takes 2~3 weeks for most cases, and 3~4 weeks for gamma PNA or special labeling.
