Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.
Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of a target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His)6-PNA chimera exhibits strong binding to chelated Ni2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His)6-PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni(2+)-NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touchstones of any nucleic acid purification scheme. We show that the specificity of the (His)6-PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His)6-PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure.
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D006639:Histidine; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009532:Nickel; D009571:Nitrilotriacetic Acid; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000302:isolation & purification* / Q000378:metabolism; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012313:RNA; D009532:Nickel; D009571:Nitrilotriacetic Acid