Peptide Nucleic Acids (PNAs) for Nucleic Acid Purification
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What is the nucleic acid purification?
A crucial step in molecular biology is nucleic acid purification, which entails extracting RNA or DNA from biological substances. Cloning, sequencing, diagnostics, and gene expression studies are just a few of the many uses that rely on this purification. For downstream applications to yield accurate results, it is imperative that purified nucleic acids are of high quality and devoid of impurities such as proteins, lipids, and other nucleic acids. The experiments that demand a lot of material need purified nucleic acids in large quantities. Popular approaches to purifying nucleic acids include phenol-chloroform extraction, columns based on silica, purification using magnetic beads, the alkaline lysis technique, and precipitation. Peptide nucleic acid (PNA) is a man-made polymer that stands in for DNA or RNA; it has several uses in molecular biology, one of which is the purification of nucleic acids. PNAs are useful for separating nucleic acids from complicated biological samples due to their one-of-a-kind characteristics. They have the potential to be engineered into capture probes that bind to specific nucleic acid sequences inside a sample. Once bound, there are a number of ways to separate the complexes, such as using magnetic beads or filtering. To separate PNAs from other, non-target nucleic acids, hybridization-based purification uses PNAs to preferentially hybridize with complementary nucleic acid targets. Because of its high level of specificity, it is possible to isolate individual sequences from very complicated combinations. It is possible to construct affinity purification systems by affixing PNAs to solid supports or beads. Because of this, nucleic acid samples may be selectively extracted depending on their sequence complementarity.
PNA for purification of human genomic DNA
With complementary nucleic acids, short PNAs (9-mer and 10-mer) made entirely of pyrimidine residues can construct thermostable triplexes. Due to their relative brevity, big nucleic acids will contain their target sequence. Because of this, PNAs like these have the potential to be utilized in the purification of big nucleic acids as general capture probes. In low-salt concentrations, PNA can disrupt native nucleic acid structures, allowing it to hybridize and purify nucleic acids. Because of their strong binding affinities, PNAs are useful for the purification of some nucleic acids. There may be uses for this technique in cancer studies. For the purpose of nucleic acid purification, Orum's team developed an oligo-histidine PNA containing six histidine residues. The Ni(II) ions may be tightly bound to by the 6×His-PNA that was created. It was possible to purify the 6×His-PNA/DNA complex using metal ion affinity chromatography by employing a Ni(II)-NTA (nitrilotriacetic acid) resin. Using numerous 6×His-PNA probes, they further proved that long RNAs (2224 nucleotides) may be efficiently collected.
PNA for purification of environmental DNA and RNA
Subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts were tested using bis-peptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides as affinity purification reagents. A PNA clamp restored a greater amount of target DNA compared to PNA or DNA oligomers under low-salt hybridization settings (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]). In most cases, using a low-salt extraction-hybridization buffer and an abundance of nontarget DNA improved the performance of PNA clamps and oligomers. However, in a very salty environment (200 mM NaPO4, 100 mM disodium EDTA, and 0.5% SDS), the DNA oligomer showed far higher capture efficiency than the PNA clamp and PNA oligomer. The particular probe, solution background, and salt condition dictated the recovery and detection efficiency, which were typically greater than 20% for target DNA concentrations of 100 pg or more. In a high-salt buffer, the DNA probe may uncover targets as small as 100 fg (830 zM [1 zM = 10−21 M]). Both bis-PNA and PNA oligomer failed to attain the same absolute detection limit when no foreign DNA was present, even when subjected to a more favorable low-salt hybridization environment. While the DNA oligomer offered less sensitivity to absolute purification and detection (830 zM) in the presence of a soil background, both PNA probes demonstrated superior performance. In a high-salt buffer, DNA capture probes performed better than PNA probes and clamp probes in a variety of environmental samples. The quantitative results for DNA target recovery were similar to those for 16S rRNA recovery from environmental samples; however, PNA probes detected more frequently from samples with a higher concentration of nontarget DNA and RNA, and the DNA oligomer produced more positive results than the bis-PNA or PNA oligomer. Based on the significant interactions between probe type and environmental sample, the most effective capture technique is determined by the specific sample type, target (DNA or RNA), and detection objective, as well as the background nucleic acid concentration.
The average efficiency of capture and elution throughout varying hybridization times. (Chandler D P., et al., 2000)
The average efficiency of capture and elution across various chemical backgrounds. (Chandler D P., et al., 2000)
PNA for sequence-specific oligonucleotide purification
Because of its enhanced stability, PNA can easily hybridize to double-stranded DNA (dsDNA) under nondenaturing circumstances by forming stable, sequence-specific connections with dsDNA through a strand-invasion process. Since plasmids and PCR products may be labeled without completely separating the dsDNA strands, this is a significant characteristic of PNA in relation to bioseparations. One kind of PNA peptide is the "PNA amphiphile (PNAA)" which is covalently bound to alkane chains. To achieve micellization and water solubility, charged amino acids are joined to the PNA peptide headgroup of the PNAA. This is necessary since both the PNA peptide and the attached alkanes are nonpolar.
According to one research, sequence-specific nucleic acid separations are made possible by the peptide nucleic acid amphiphile C12-agtgatctac-(Glu)4, which binds to a corresponding DNA sequence with great specificity and selectivity while maintaining its stability throughout the HIC chromatographic process. It is not necessary to boil or wash the material to remove partly bound DNA; incubation periods on the range of minutes can produce almost quantitative DNA recovery. For short sequence lengths, DNA contaminants may be effectively resolved from PNAA/DNA duplex peaks. However, in order to maintain equivalent capacity factors for longer sequences, triplex formation or multiple binding sites on the target DNA may be required. Continuous work is being done to exploit the strand-invasion capabilities of PNA to target samples of double-stranded DNA, such as plasmids and PCR products.
The structure of PNA C12-agtgatctac-(Glu)4. (Vernille J P., et al., 2004)
Significance of PNA for nucleic acid purification
High specificity and affinity: Because of their distinct backbone structure, PNAs show robust and highly specific hybridization with complementary nucleic acids. As a result, the quality of the isolated nucleic acids is improved and the chances of non-specific binding are decreased. Even in complicated biological mixtures, PNAs can reliably capture target nucleic acids because they create more stable complexes than traditional DNA/RNA.
Durability: PNAs have a major benefit during purification operations as they are not degraded enzymatically by nucleases. Because of their stability, the nucleic acids may be handled for longer periods of time without worrying about damaging them.
Versatility: PNAs can be engineered to target diverse nucleic acid sequences, encompassing mRNA, DNA, and particular mutations. This adaptability renders them appropriate for many applications in research and diagnosis. PNAs are applicable in several purification techniques, including affinity chromatography, hybridization-based capture, and magnetic bead-based separation, hence broadening their utility across numerous processes.
Improved sensitivity: The increased binding affinity of PNAs facilitates the purification and detection of low-abundance nucleic acid targets, essential for applications such as early disease diagnosis and environmental monitoring.
Ready for further analysis: Nucleic acids purified by PNAs often exhibit superior quality and purity, rendering them optimal for further applications including quantitative PCR, sequencing, and microarray research. This results in enhanced reliability and reproducibility in study outcomes.
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References
- Orum H., et al., Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection, BioTechniques, 1995, 19(3): 472-480.
- Seeger C., et al., PNA-mediated purification of PCR amplifiable human genomic DNA from whole blood, Biotechniques, 1997, 23(3): 512-517.
- Chandler D P., et al., Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps, Applied and Environmental Microbiology, 2000, 66(8): 3438-3445.
- Vernille J P., et al., Sequence-specific oligonucleotide purification using peptide nucleic acid amphiphiles in hydrophobic interaction chromatography, Biotechnology progress, 2004, 20(6): 1776-1782.
