Targeting a KRAS i-motif forming sequence by unmodified and gamma-modified peptide nucleic acid oligomers.

Growing interest in i-motif DNA as a transcriptional regulatory element motivates development of synthetic molecules capable of targeting these structures. In this study, we designed unmodified peptide nucleic acid (PNA) and gamma-modified PNA (γPNA) oligomers complementary to an i-motif forming sequence derived from the promoter of the KRAS oncogene. Biophysical techniques such as circular dichroism (CD) spectroscopy, CD melting, and fluorescence spectroscopy demonstrated the successful invasion of the i-motif by PNA and γPNA. Both PNA and γPNA showed very strong binding to the target sequence with high thermal stability of the resulting heteroduplexes. Interestingly fluorescence and CD experiments indicated formation of an intermolecular i-motif structure via the overhangs of target-probe heteroduplexes formed by PNA/γPNA invasion of the intramolecular i-motif. Targeting promoter i-motif forming sequences with high-affinity oligonucleotide mimics like γPNAs may represent a new approach for inhibiting KRAS transcription, thereby representing a potentially useful anti-cancer strategy.

Applications of PNA-laden nanoparticles for hematological disorders.

Safe and efficient genome editing has been an unmitigated goal for biomedical researchers since its inception. The most prevalent strategy for gene editing is the use of engineered nucleases that induce DNA damage and take advantage of cellular DNA repair machinery. This includes meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems. However, the clinical viability of these nucleases is marred by their off-target cleavage activity (≥ 50% in RNA-guided endonucleases). In addition, in vivo applications of CRISPR require systemic administration of Cas9 protein, mRNA, or DNA, which presents a significant delivery challenge. The development of nucleic acid probes that can recognize specific double-stranded DNA (dsDNA) regions and activate endogenous DNA repair machinery holds great promise for gene editing applications. Triplex-forming oligonucleotides (TFOs), which were introduced more than 25 years ago, are among the most extensively studied oligomeric dsDNA-targeting agents. TFOs bind duplex DNA to create a distorted helical structure, which can stimulate DNA repair and the exchange of a nearby mutated region-otherwise leading to an undesired phenotype-for a short single-stranded donor DNA that contains the corrective nucleotide sequence. Recombination can be induced within several hundred base-pairs of the TFO binding site and has been shown to depend on triplex-induced initiation of the nucleotide excision repair pathway and engagement of the homology-dependent repair pathway. Since TFOs do not possess any direct nuclease activity, their off-target effects are minimal when compared to engineered nucleases. This review comprehensively covers the advances made in peptide nucleic acid-based TFOs for site-specific gene editing and their therapeutic applications.

A rapid bioanalytical tool for detection of sequence-specific circular DNA and mitochondrial DNA point mutations.

Mutations in mitochondrial DNA (mtDNA) have been an essential cause of numerous diseases, making their identification critically important. The majority of mtDNA screening techniques require polymerase chain reaction (PCR) amplification, enzymatic digestion, and denaturation procedures, which are laborious and costly. Herein, we developed a sensitive PCR-free electrokinetic-based sensor combined with a customized bis-peptide nucleic acid (bis-PNA) and gamma-PNA (γ-PNA) probes immobilized on beads, for the detection of mtDNA point mutations and sequence-specific supercoiled plasmid DNA at the picomolar range. The probes are capable of invading the double-stranded circular DNA and forming a stable triplex structure. Thus, this method can significantly reduce the sample preparation and omit the PCR amplification steps prior to sensing. Further, this bioanalytical tool can open up a new paradigm in clinical settings for the screening of double-stranded circular nucleic acids with a single-base mismatch specificity in a rapid and sensitive manner.

Simultaneous genotyping of multiple somatic mutations by using a clamping PNA and PNA detection probes.

It has been very difficult to detect and quantify multiple somatic mutations simultaneously in single-tube qPCR. Here, a novel method for simultaneous detection of multiple mutations and a melting curve analysis was developed by using clamping PNA and detection PNA probes. Each PNA probe was designed to have a specific melting temperature by the introduction of γ-PNA monomer. This technique was successfully applied to the detection of six genotypes in two different mutations of K-RAS at the same time. Such simultaneous analysis of an amplified curve and a melting curve in qPCR can be widely used for the early diagnosis of cancer and determining the prognosis of drug treatments.

Effect of chirality in gamma-PNA: PNA interaction, another piece in the picture.

Modification of the PNA backbone can be used to broaden their utility by introducing new functional groups. In particular, gamma-modified PNA have been found to be quite effective in a number of applications, and exhibit particularly high DNA binding affinity. The introduction of one side chain imply that the achiral backbone of PNA becomes chiral, and binding properties depend on the stereochemistry. A new paper on gamma-modified PNA by Ly and co-workers complete the existing knowledge by displaying that in binding to complementary PNA stereochemical orthogonality can be demonstrated. This opens the way to the exploitation of stereochemical features in diagnostic assays and in nanofabrication.