Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Peptide nucleic acids (PNA) offer high affinity and specificity for complementary nucleic acids.
• Amino acid attachment during PNA synthesis allows for functional modifications.
• Existing nucleic acid purification methods require optimization for specificity and efficiency.

Purpose of the Study:

• To develop a rapid and efficient method for sequence-specific purification of target nucleic acids.
• To create a novel oligohistidine-peptide nucleic acid (oligohistidine-PNA) chimera for purification applications.
• To leverage PNA's hybridization properties and amino acid conjugations for affinity-based purification.

Main Methods:

• Synthesis of oligohistidine-PNA chimeras.
• Utilizing the (His)6-PNA chimera's binding to chelated Ni2+ ions.
• Employing metal ion affinity chromatography with Ni(2+)-NTA resin for purification.
• Assessing purification specificity for single-nucleotide differences and efficiency for large RNAs.

Main Results:

• The (His)6-PNA chimera binds strongly to Ni2+ ions without affecting hybridization properties.
• Purification of (His)6-PNA/DNA complexes was achieved using Ni(2+)-NTA affinity chromatography.
• High specificity was demonstrated by purifying oligonucleotides differing by a single nucleotide.
• Efficient capture of large RNAs (2224 nucleotides) was achieved using multiple (His)6-PNA probes.
• Purification of target oligonucleotides within intramolecular stem-loop structures was demonstrated.

Conclusions:

• The oligohistidine-PNA chimera provides a robust platform for rapid, sequence-specific nucleic acid purification.
• Metal ion affinity chromatography is an effective strategy for purifying PNA-nucleic acid complexes.
• The method offers high specificity and efficiency, applicable to various nucleic acid targets, including those in complex structures.