Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA.

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design. We developed novel Ps-TFOs containing 2'-O,4'-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2 , 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.

Therapeutic application of sequence-specific binding molecules for novel genome editing tools.

Genome editing has been expected to widely increase the available treatment options for various diseases and permit pharmaceutical interventions in previously untreatable conditions. The availability of genome editing tools was dramatically increased by the development of the CRISPR-Cas9 system. However, a number of issues limit the use of the CRISPR-Cas9 system and other gene-editing tools in the clinical treatment of diseases. This review summarized the history and types of genome editing tools and limitations of their use. In addition, the study addressed several next-generation technologies aiming to overcome the limitations of current gene therapy protocols in an effort to accelerate the clinical development of potential treatment options. This review has provided an extensive foundation of the current state of genome editing technology and its clinical development. This review also indicate that the study additionally highlighted the need for multidisciplinary approaches to overcome current bottlenecks in the development of genome editing.

Selective Photo-Crosslinking Detection of Methylated Cytosine in DNA Duplex Aided by a Cationic Comb-Type Copolymer.

In the process of cell development and differentiation, C-5-methylation of cytosine (5-methylcytosine: 5-mC) in genome DNA is an important transcriptional regulator that switches between differentiated and undifferentiated states. Further, abnormal DNA methylations are often present in tumor suppressor genes and are associated with many diseases. Therefore, 5-mC detection technology is an important tool in the most exciting fields of molecular biology and diagnosing diseases such as cancers. In this study, we found a novel photo-crosslinking property of psoralen-conjugated oligonucleotide (Ps-Oligo) to the double-stranded DNA (ds-DNA) containing 5-mC in the presence of a cationic comb-type copolymer, poly(allylamine)-graft-dextran (PAA-g-Dex). Photo-crosslinking efficiency of Ps-Oligo to 5-mC in ds-DNA was markedly enhanced in the presence of PAA-g-Dex, permitting 5-mC-targeted crosslinking. We believe that the combination of PAA-g-Dex and Ps-Oligo will be an effective tool for detecting 5-mC in genomic DNA.