as Photo-crosslinkable Gene Targeting Tools

Several psoralen-conjugated oligonucleotides (Ps-Oligos) have been developed as photo-crosslinkable oligonucleotides targeting DNA or RNA. To avoid potential off-target effects, it is important to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA or RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far. In this study, we evaluated the photo-cross-linking properties of two Ps-Oligos, 5’-Ps-Oligo and a novel Ps-Oligo containing 2’- O -{[(4,5’,8-trimethylpsoralen)-4’-ylmethoxy]ethylaminocarbonyl}adenosine (APs2-Oligo). Notably, 5’-Ps-Oligo preferentially crosslinked with DNA, whereas APs2-Oligo preferentially crosslinked with RNA. These results demonstrate the in-teresting crosslinking properties of Ps-Oligos, which will provide useful information for the molecular design of novel Ps-Oligos in future studies. Our results suggest that the RNA-selective crosslinking property of APs2-Oligo is not caused by structural differences in the target nucleobase. From the structural simulation analysis, it is proposed that APs2-Oligo forms a more suitable crosslinking intermediate with the target RNA upon duplex formation. This study provides a valuable knowledge for the molecular design of novel Ps-Oligos targeting DNA (5’-Ps-Oligo) or RNA (APs2-Oligo) in the future. Further investigations are ongoing by our research


Introduction
Psoralen derivatives are used as dermal photosensitizing molecules for the treatment of various skin pigmentation disorders. [1][2][3] These compounds are also known as DNA intercalators and can form covalent bonds via the [2+2] cycloaddition reaction with pyrimidine bases upon ultraviolet (UV) irradiation. 4,5) Therefore, psoralens have been applied as one of DNA intercrosslinking 6,7) agents in various research fields. [8][9][10] Several research groups have developed psoralen-conjugated oligonucleotides (Ps-Oligos) targeting DNA or RNA. These Ps-Oligos can be used to form crosslinkages in a sequence-specific manner via duplex formation. Some of Ps-Oligos have been used as antisense oligonucleotides and suppressed tumor growth by crosslinking with target mRNA and interfering with mRNA translation. [11][12][13][14] In contrast, we have developed Ps-Oligos, which can distinguish a point mutation (from a purine base to a pyrimidine base) in DNA in a sequence-specific manner based on the photo-crosslinking strategy. 15,16) These Ps-Oligos can be used for the detection of oncogenes, such as ras, egfr, myc, and erb-B, which are wellknown causes of tumorigenesis. 17,18) The selective detection method for these oncogenes may provide valuable insight into the development mechanisms of these diseases. Thus, Ps-Oligos are expected to be a genome-targeting technology.
As described above, Ps-Oligos are widely used as photo-crosslinkable oligonucleotides that target DNA and RNA. Therefore, to avoid potential off-target effects, it is essential to investigate the selective photo-crosslinking reactivity of Ps-Oligos to DNA and RNA. However, the selectivity of these Ps-Oligos has not been reported in detail thus far, and information on the relationship between the Ps-Oligo structure and its target selectivity is limited. This study aims to assess the photo-crosslinking property of 5'-Ps-Oligo (Fig. 1A), which possesses a psoralen moiety at its 5'-end,

Synthesis of APs2-Oligo
The preparation of APs2-Oligo was started with the synthesis of 1). We decided to introduce psoralen via carbamate linkage, since it is synthetically easier to prepare than the previously reported 2'-O-psoralenylmethoxyethyladenosine. 12) First, the alcohols at the 5' and 3' positions were simultaneously protected by disiloxane 2 according to a procedure reported in literature to give compound 3. 19) Protected compound 3 was treated with carbonyldiimidazole to generate reactive

Crosslinking Behavior of 5'-Ps-Oligo and APs2-Oligo
The photo-crosslinking behaviors of 5'-Ps-Oligo and APs2-Oligo were evaluated and the results were compared (Fig. 1). The fidelity in target sequence recognition by oligonucleotides is an important factor that influences photo-crosslinking efficiency, which is attributed to the difference in the thermal stability of the target hybrids. To properly evaluate the photo-crosslinking selectivity of 5'-Ps-Oligo toward DNA or RNA, two sequences were prepared for target DNA and RNA, as shown in Fig. 1. These sequences showed slightly higher Tm value (Supplementary Subsequently, the photo-crosslinking efficiencies of 5'-Ps-Oligo and APs2-Oligo were evaluated using denaturing polyacrylamide gel electrophoresis (denaturing PAGE). UV irradiation was conducted for a maximum of 120 s, and samples were collected at different time steps (0, 1, 5, 10, 20, 30, 60, and 120 s). Consequently, 5'-Ps-Oligo was selectively crosslinked with DNA ( Fig. 2A) but not efficiently with RNA (Fig. 2B). The crosslinking efficiency of 5'-Ps-Oligo with DNA was 40% higher than that with RNA. On the other hand, APs2-Oligo crosslinked with both DNA (Fig. 3A) and RNA (Fig. 3B), and interestingly, the crosslinking efficiency of APs2-Oligo with RNA was higher than that with DNA (Fig. 3C).

The Difference in Duplex Structure May Affect Target Preference
To determine whether the RNA selectivity of APs2-Oligo in a crosslinking reaction is influenced by differences in the nucleobase structure (DNA: thymine, RNA: uracil), a photo-crosslinking experiment was conducted using DNA in which the target thymine was replaced with uracil (DNA-U). Interestingly, the crosslinking efficiencies of APs2-Oligo with DNA and DNA-U were nearly the same (Fig. 4).

Chemical and Pharmaceutical Bulletin Advance Publication
These results suggest that the preferential crosslinking of APs2-Oligo to RNA is not caused by structural differences in the target nucleobase. Based on these results, we propose that certain structural differences in the duplex (DNA duplex generally forms B-form double helix, while DNA-RNA duplex adopts the A-form for both strands) 21-23) may affect the target preference of APs2-Oligo during photo-crosslinking. We conducted structural simulations of duplex models for both 5'-Ps-Oligo ( Supplementary Fig. S6) and APs2-Oligo ( Supplementary Fig. S7). The psoralen moiety of 5'-Ps-Oligo stacks with target thymine in DNA models, while the psoralen of 5'-Ps-Oligo is away from the target uracil in RNA models. This is consid-

Photo-crosslinking Reaction
Equimolar mixtures of TAMRA labeled 5'-Ps-Oligo or APs2-Oligo and their complementary oligonucleotides were prepared in a similar manner to the samples prepared for UV-melting profile measurements. After incubation, the reaction mixtures were UV-irradiated for 0-120 s using a UV-LED irradiator (OMRON ZUV-C30H, 365 nm), followed by analysis using denaturing PAGE (20% polyacrylamide/7 M urea/25% formamide). The crosslinking efficiencies were quantified by measuring TAMRA fluorescence.