Synthesis of a Pseudocytidine Nucleoside to Form a Stable and Selective Base Pair with Iso-guanosine in RNA

Abstract

Non-natural base pair formation provides insight into new functions of nucleic acids. Therefore, various artificial base pairs have been developed in both DNA and RNA. In this work, we successfully synthesized pseudocytidine from commercially available pseudouridine to form base pairs with isoguanine, also known as 2-OH-adenine, in RNA. Measurement of the melting temperature with the base pair incorporated at the center of a 13-mer RNA showed the highest value for the ψ-rC and iso-rG (2-OH-rA) base pair. This base pair formation exhibited a high melting temperature regardless of whether it was incorporated into the pyrimidine or purine strand, indicating that it can form a stable and selective duplex RNA.

The rational design and incorporation of unique base pair formations into RNA duplexes have emerged as powerful strategies to overcome the inherent limitations of natural nucleobase pairing in nucleic acid engineering. While Watson–Crick base pairs serve as the basis for the structural integrity and informational fidelity of RNA, their strict complementarity restricts the programmability and chemical diversity required for advanced RNA-based applications. − Expanding the chemical and functional landscape of RNA through the introduction of synthetic base pairs has therefore become a central objective in the development of next-generation nucleic acid technologies. − Artificial base pairs, engineered to form orthogonal and sequence-specific interactions, can enhance the thermodynamic stability of RNA duplexes, increase structural versatility, and enable the recognition of epigenetically or synthetically modified nucleotides. These systems not only are instrumental in probing RNA folding and function but also hold promise for therapeutic modalities, such as aptamer-based targeting, siRNA stabilization, and programmable riboregulators. Furthermore, these unique base pairs can serve as chemical handles for site-selective functionalization, allowing for the precise positioning of fluorophores, cross-linkers, or reactive groups within RNA scaffolds, facilitating structural studies, molecular imaging, and chemical biology applications. −

Recently, we successfully developed and reported a pseudo-dC (ψ-dC) that can form selective and stable base pairs with iso-dG (2-OH-dA) in DNA. In this study, we aimed to synthesize the ribonucleoside form of pseudocytidine (ψ-rC) (1) and evaluate its base pairing properties (Figure ). In parallel, the 2-position oxidized form of adenine (2-OH-rA, i.e., iso-rG) has been identified in cellular RNA, and its potential involvement in disease-related processes has been suggested. − Therefore, the development of artificial molecules capable of selectively recognizing this oxidized nucleobase is of significant interest in the context of RNA chemical biology and diagnostics. −

1.

Structure of ψ-rC and iso-rG in this study.

The synthesis of the ψ-rC phosphoramidite derivative (7) is depicted in Scheme . Using commercially available pseudouridine (ψ-U) (2) as a starting material, the hydroxyl groups at the sugar moiety were protected with TBS groups and the nitrogen atom at position 1 of nucleobase was protected with a benzyl group to obtain compound 3. Then, compound 4 was obtained by activation of the carbonyl at position 4 of 3 with 2,4,6-triisopropylbenzenesulfonyl chloride and treatment with a 28% aqueous ammonia solution in a good yield. The amino group was protected with a benzoyl group to apply to an automated synthesizer, and it was converted into triol compound 5. The solubility of the triol compound was poor, which complicated purification and resulted in a very low yield; this remains a challenge to be addressed in future work. However, we decided to proceed with the next reaction. We attempted to use Et₂SiCl₂ to protect triol compound 5 at the 3′- and 5′-positions, which gave a yield of less than 50%. This result is also thought to be due to the poor solubility of the compound. After DMTr protection of triol compound 5 at the 5′-position, the 2′-position was protected with a TBDMS group, followed by attempted phosphitylation. However, no reaction progress was observed. Therefore, the hydroxyl groups at the 5′- and 2′-positions were protected with the DMTr group and TOM groups with yields of 76% and 30%, respectively, to isolate and purify target compound 6. Additionally, compound 6′, bearing a TOM-protected 3′-hydroxyl group, was isolated in 18% yield. Here, the compound with the hydroxyl group at the 3′-position protected by a TOM group was also obtained in nearly the same yield. Finally, the phosphoramidite derivative, compound 7, was obtained for use in the oligonucleotide synthesis. Despite the extremely poor solubility of all ψ-rC derivatives in organic solvents in this synthesis, which complicated purification, their conversion to the corresponding phosphoramidite derivatives was successfully accomplished. While the specific identity of the impurities is unknown, we proceeded with RNA synthesis to assess whether they would have any practical effect. The RNA, oligoribonucleotide (ORN), syntheses were carried out by Japan Bio Services Co., Ltd. After incorporating this compound into the center of the 13-mer RNA with a coupling time of 25 min, compared to the typical 10 min coupling time for natural nucleosides, they were produced using general purification methods, and the purities and structures of synthesized ORN1 (5′-r­(CUUUCUXCUCCUU)-3′, X = ψ-rC, MW = 3912.24) and ORN2 (5′-r­(AAGGAGYAGAAAG)-3′, X = ψ-rC, MW = 4273.69) were confirmed by LC/ESI-MS instrument in negative ion mode. The observed mass values were 3912.35 and 4273.90, respectively (Figure ).

1. Synthesis of the ψ-rC Phosphoramidite Derivative.

2.

MS analysis results of synthesized (A) ORN1 and (B) ORN2 containing ψ-rC.

Next, the melting temperature (T _m) was measured using synthesized ORNs. The UV melting curves are shown in Figure , where ORN1 or ORN2 containing ψ-rC was mixed with complementary ORNs containing rG, rA, rC, U, or iso-rG at the corresponding position. Then, the absorbance at 260 nm was plotted as a function of temperature.

3.

UV melting curves of duplex RNA formation between ORN1 and ORN2. UV melting profiles were measured using a solution containing each ORN strand at a concentration of 3 μM in 100 mM NaCl, 7.5 mM MgCl₂, and 5 mM sodium phosphate buffer at pH 6.9, which showed (A) ORN1 and (B) ORN2 containing ψ-rC.

In the sequence containing ψ-rC on pyrimidine strand ORN1, low melting temperatures were observed when pyrimidine nucleobases, U and C, were present at the complementary position in the purine strand of ORN2 (Figure A, gray and yellow lines for rU and rC, respectively). In contrast, when purine nucleobases, G, A, and iso-G, were placed opposite ψ-rC, higher melting temperatures were observed, with the highest melting temperature recorded for iso-rG in ORN2 (Figure A, blue, orange, and red lines for rG, rA, and iso-rG, respectively). A similar melting profile was observed in purine strand ORN2 containing ψ-rC, where the ψ-rC and iso-rG base pair formation exhibited the highest melting temperature in combination with natural bases (Figure B, red line). These results indicate that ψ-rC exhibits a high recognition ability toward iso-rG in the RNA duplex.

The T _m values are summarized in Table , together with those of canonical base pairs for comparison. In the case of ORN1 containing ψ-rC, it was 62.6 °C with respect to the iso-rG nucleoside in the complementary ORN2 strand. Under these conditions, the T _m values were 49.6 and 50.3 °C for purine nucleosides rG and rA, respectively, in complementary ORN2, whereas those for pyrimidine nucleosides U and rC were 38.7 and 44.6 °C, respectively. The corresponding natural base pairs, rC/rG and U/rA, showed T _m values of 63.1 and 56.5 °C, respectively. By a comparison of these values, it was found that the base pair formation between ψ-rC in ORN1 and iso-rG in ORN2 was stable and effective within the RNA duplex. Similarly, when ψ-rC was present in ORN2, the base pair formation with iso-rG in ORN1 showed a T _m value of 62.2 °C, which was also comparable to those of the natural base pairs, rG/rC and rA/U, which showed T _m values of 61.7 and 56.3 °C, respectively. Furthermore, when the melting temperature of ψ-rC with iso-rG was compared to those of ψ-rC with other bases, a difference of more than 10 °C was observed, demonstrating the high selectivity of ψ-rC/iso-rG base pairing.

1. Melting Temperatures of Duplex RNA Formation .

ORN1	ORN2	T m (°C)	ORN1	ORN2	T m (°C)
rC	rG	63.1	rG	rC	61.7
U	rA	56.5	rA	U	56.3
ψ-rC	rG	49.6	rG	ψ-rC	49.1
ψ-rC	rA	50.3	rA	ψ-rC	50.4
ψ-rC	U	38.7	U	ψ-rC	41.0
ψ-rC	rC	44.6	rC	ψ-rC	45.6
ψ-rC	iso-rG	62.6	iso-rG	ψ-rC	62.2

^aUV melting profiles were measured using a solution containing each ORN strand at a concentration of 3 μM in 100 mM NaCl, 7.5 mM MgCl₂, and 5 mM sodium phosphate buffer at pH 6.9. Data were analyzed using the melting curve processing program. T _m values are the average of three or more different experiments and are within ±0.5 °C.

Next, the thermodynamic parameters associated with the formation of the RNA duplexes were evaluated (Figure S1 and Table ). These results indicate that the ψ-rC/iso-rG base pair (Table , entries 1 and 2) exhibits thermodynamic properties more closely resembling those of the rC/rG base pair (Table , entries 3 and 4) than the U/rA base pair (Table , entries 5 and 6), suggesting the potential formation of three hydrogen bonds.

2. Thermodynamic Parameters of Duplex RNA Formation .

entry	X in ORN1	Y in ORN2	ΔH° (kcal/mol)	ΔS° (cal K–1 mol–1)	ΔG°310 K (kcal/mol)
1	ψ-rC	iso-rG	–123.47	–339.86	–18.06
2	iso-rG	ψ-rC	–101.32	–274.18	–16.29
3	rC	rG	–123.38	–340.51	–17.77
4	rG	rC	–101.07	–272.62	–16.52
5	U	rA	–121.91	–341.76	–15.92
6	rA	U	–95.38	–261.49	–14.28

^aUV melting profiles were measured using a solution containing each ORN strand at a concentration of 1–4 μM in 100 mM NaCl, 7.5 mM MgCl₂, and 5 mM sodium phosphate buffer at pH 6.9. The thermodynamic parameters of the complex between ORN1 and ORN2 were assessed by van’t Hoff plots with four data points and the curve-fitting method.

In conclusion, we successfully achieved the first chemical synthesis of a novel artificial nucleoside, ψ-rC, derived from ψ-U as the starting material. Although the synthesized compounds exhibited poor solubility, we successfully incorporated the target compound into ORNs. In base pairing studies, a high melting temperature was observed when the iso-rG was exhibited opposite to ψ-rC, indicating the formation of a highly stable and selective base pair. While further optimization of the synthetic route is necessary, we are currently exploring potential applications utilizing this novel base pair between the ψ-rC and iso-rG base pair.

The data underlying this study are available in the published article and its Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.orglett.5c03060.

• General methods, synthesis, NMR spectra, and van’t Hoff plots (PDF)

The authors declare no competing financial interest.