8-Pyrenylvinyl Adenine Controls Reversible Duplex Formation

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8‑Pyrenylvinyl Adenine Controls Reversible Duplex Formation between Serinol Nucleic Acid and RNA by [2 + 2] Photocycloaddition Keiji Murayama, Yuuhei Yamano, and Hiroyuki Asanuma* Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan

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S Supporting Information *

ABSTRACT: Photocontrol of duplex formation between the totally artificial serinol nucleic acid (SNA) and target RNA was made possible using a photoresponsive nucleobase 8-pyrenylvinyl adenine (PVA). PVA residues in SNA can be induced to undergo intrastrand [2 + 2] photocycloaddition by 455 nm light. Effective cycloreversion of the PVA photodimer results from irradiation with 340 nm light. These reactions occurred in high yield, rapidly, selectively, and reversibly. When the PVA-SNA/ RNA duplex was irradiated with 455 nm light, almost complete dissociation of the duplex was attained, and 340 nm light restored duplex formation by cycloreversion. This is the first example of use of photocycloaddition and cycloreversion to photoregulate canonical duplex formation and dissociation reversibly at constant temperature. Thus, SNA bearing PVA residues have potential for use in photocontrollable biological tools targeting endogenous RNAs in cells as well as photodriven SNA machines.

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eno nucleic acids (XNAs), synthetic analogues of DNA and RNA carrying scaffolds different from natural Dribose, impart new functions and high nuclease resistance in biological applications of nucleic acids.1−6 Recently, pure XNAs that do not include any natural nucleotides have attracted attention as a new nanomaterial for nanomachines and nanoarchitectures.7−10 Several acyclic XNAs bearing phosphodiester linkages have also been synthesized.11−15 Our group has also developed the acyclic XNAs SNA16 and LaTNA,17 which can hybridize with DNA and particularly with RNA. Notably, SNA could be applied to various biological tools such as antisense-mediated exon skipping,18 anti-miRNA oligonucleotides,19 and detection of mRNA in cells.20,21 Incorporation of additional functionalities to SNA would further expand the scope of its application. A useful function is stimulus-responsivity in which a function is activated in response to an external stimulus such as pH, heat, or metal ion binding.22−25 Among potential external stimuli, light is an ideal trigger because spatiotemporal control is possible without contaminating the solution.26−31 For reversible photoregulation, photochromic molecules that isomerize upon irradiation have been developed.32−34 Wagenknecht and Jaschke designed photoresponsive nucleobases involving diarylethene.35,36 Guanine analogues carrying arylvinyl moieties synthesized by Ogasawara and coworkers © 2019 American Chemical Society

Figure 1. (a) Chemical structures of SNA and 8-pyrenylvinyl adenine (PVA). (b) Scheme and illustration of reversible photoregulation of SNA/RNA duplex formation via intrastrand photo-cross-linking of PV As in the SNA strand. Irradiation with 455 nm blue light induces dissociation of the duplex through [2 + 2] photocycloaddition, whereas irradiation with 340 nm UV light causes reformation of the duplex via cycloreversion.

have also been used to reversibly photoregulate DNA duplex or G-quadruplex formation.37,38 Our group has developed photoresponsive DNA and RNA by introducing azobenzene via D-threoninol or other acyclic diols into oligonucleotides.39 Despite the great applicability of XNAs, photoregulation of the XNA duplex has not yet been achieved. We had attempted to photoregulate SNA using azobenzene; however, insertion of azobenzene severely destabilized the duplex even in the trans-form (Figure S1). To develop a photoregulatable modification of SNA, we designed 8pyrenylvinyl adenine (PVA), which should not interfere with canonical Watson−Crick base pairing with thymine or uracil Received: March 26, 2019 Published: May 22, 2019 9485

DOI: 10.1021/jacs.9b03267 J. Am. Chem. Soc. 2019, 141, 9485−9489

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Journal of the American Chemical Society Table 1. Sequences of Synthesized SNAs and Melting Temperatures of Duplexes with Complementary RNA SNA-N SNA-P2P SNA-P1P SNA-P0P SNA-P SNA-P0P-Q

sequence (SNA)

Tm with RNAa

(S)-GCATCAGT-(R) (S)-GCPVATCPVAGT-(R) (S)-TCGPVATPVAGA-(R) (S)-GCTPVAPVATGC-(R) (S)-GCTPVAATGC-(R) (S)-QGCTPVAPVATGC-(R)

35.0b −c −c 35.1 30.0 28.0d

a

RNA sequences are listed in Table S1. bData were obtained from ref 16. cNo sigmoidal curve was observed. dCalculated from melting curves of fluorescence emission (Figure S11d).

Figure 3. (a) Absorption spectra, (b) melting profile, and (c) CD spectra of SNA-P0P/RNA before (black lines) and after irradiation with 455 nm light (green lines) and 340 nm light (blue lines). Energy minimized structures of (d) duplex SNA-P0P/RNA and (e) single stranded SNA-P0P containing PVA photodimer. SNA, RNA, and PVAs are presented as cyan, black, and magenta, respectively.

photocycloaddition reaction has not yet been applied to regulate canonical duplex formation and dissociation at constant temperature.46 The route for synthesis of the SNA−PVA phosphoramidite monomer is shown in Scheme S1. Three SNA sequences with two PVA residues at different positions (SNA-P2P, SNA-P1P, and SNA-P0P) were synthesized (Table 1). For the analysis of photoreactivity in monomeric PVA, we also prepared an SNA tethering a single PVA (SNA-P). As a control, we synthesize an unmodified SNA (SNA-N). First, we irradiated single stranded SNA-P2P with 455 nm light. The absorption band at around 400 nm present before irradiation immediately decreased and almost disappeared after 2 min (Figure 2a). Simultaneously, new bands appeared at 270 and 354 nm, which correspond to absorption bands of alkylpyrene. A similar behavior was observed upon photocross-linking of styrylpyrene,40,47−49 suggesting that an intrastrand photoadduct of two PVAs was formed. Upon irradiation of the cross-linked product with 340 nm light, the initial absorption bands were restored, indicating the recovery of PVA monomers (Figure 2b). Furthermore, isosbestic points at 330 and 361 nm are indicative of selective photocycloaddition and cycloreversion reaction between the two PVA residues. HPLC analysis of SNA-P2P was performed to analyze the photoreaction (Figure 2c). The peak with retention time of 25 min present before light irradiation almost completely disappeared upon irradiation with 455 nm light, and a new single peak appeared at shorter retention time (18 min), corresponding to the intrastrand-photo-cross-linked product. Irradiation with UV light restored the initial peak accompanied

Figure 2. (a) Absorption spectra of single-stranded SNA-P2P at indicated times of irradiation with 455 nm light. (b) Absorption spectra of single-stranded SNA-P2P at photostationary state (PSS) at 455 nm and after irradiation for indicated times with 340 nm light. Irradiation was performed at 20 °C. (c) Reversed-phase HPLC profiles of single-stranded SNA-P2P before and after irradiation at 455 nm for 360 s and 340 nm for 360 s. Absorbance was monitored at 260 nm. (d) Cross-linking ratio of PVA in SNA-P2P as a function of irradiation time at 455 nm (open circles) and 340 nm (closed triangles). The cross-linking ratio was calculated from absorbance at 400 nm. (e) Fluorescence spectra before irradiation (solid line), at PSS at 455 nm (broken line), and at PSS at 340 nm (dotted line). (f) Cross-linking ratios after the indicated number of photoswitching cycles. Irradiation times were 120 s at 455 nm and 60 s at 340 nm. The reaction ratios were calculated from absorbance at 400 nm recorded after each irradiation.

(Figure 1a). Irradiation with visible blue light (455 nm) is expected to induce intrastrand cross-linking through the [2 + 2] photocycloaddition between the neighboring PVAs, distorting the SNA strand and resulting in duplex dissociation. Irradiation with 340 nm light should regenerate PVA via cycloreversion reaction and allow duplex formation (Figure 1b). Although photocycloaddition reactions have been widely utilized,40−45 to the best of our knowledge, a reversible 9486

DOI: 10.1021/jacs.9b03267 J. Am. Chem. Soc. 2019, 141, 9485−9489

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Journal of the American Chemical Society

Figure 4. Analysis of hybridization of SNA-P0P-Q and RNA-Cy3 based on the change in fluorescence change of Cy3 induced by light irradiation at 20 °C. (a) Illustration of the design of this experiment. Fluorescence from Cy3 tethered to the RNA is quenched by duplex formation with SNA containing anthraquinone. (b) Fluorescence spectra of SNA-P0P-Q/RNA-Cy3 before irradiation (black line) and after irradiation with 455 nm for 3 h (green line), followed by 340 nm for 50 min (blue line). Analysis of single-stranded RNA-Cy3 (orange line) is shown as a reference of complete dissociation, and SNA-P0P-Q/RNA-Cy3 at 5 °C (purple line) is shown as a reference for 100% duplex. (c) Cross-linking ratio after multiple photoswitching cycles calculated from fluorescence intensity.

340 nm for 720 s restored 67% of monomeric PVA in SNAP0P. Thus, intrastrand photocycloaddition and cycloreversion of PVAs in single-stranded SNAs were efficient when two PVAs were incorporated into the SNA, regardless of position. In contrast to the SNA-PnP series, irradiation of SNA-P with blue light resulted in mainly the cis-form and a small amount of interstrand cross-linking (Figure S7). For the SNA-PnP series, cis-isomerization was minimal, and intrastrand cross-linking was facilitated by the stacking interaction of the two PVAs. To determine the effect of PVA substitution on the thermal stability of a duplex, melting temperatures of SNA/RNA duplexes were determined before irradiation (Table 1, Figure S8). SNA-P/RNA was slightly destabilized compared to the control SNA-N/RNA duplex, suggesting that PVA can pair with uracil as adenine does. Unexpectedly, SNA-P2P and SNA-P1P did not form duplexes with complementary RNA. The strong stacking interaction between the two PVA residues presumably suppressed duplex formation. The adjacent PVA residues of SNA-P0P might adopt a stacked structure in the single strand that is similar to that in the duplex (Figure S9), as the Tm of SNA-P0P/RNA was similar to that of SNA-N/RNA. Next, we examined the photoregulation of SNA-P0P/RNA duplex formation. Upon irradiation with 455 nm light at 20 °C, a temperature at which all strands are involved in duplex structures, the absorption band at around 400 nm decreased with irradiation time (Figures 3a and S10a). Irradiation for 1 h resulted in almost complete cross-linking (Figure S10c). The cross-linking was slower than that observed when SNA-P0P

by a decrease in intensity of the peak at 18 min. This result demonstrates selective and reversible photocycloaddition without significant side reaction. The cross-linking ratio was plotted as a function of time (Figure 2d): 98% of PVA was cross-linked upon irradiation with 455 nm light for 180 s, whereas 340 nm light converted 90% of the photodimer into monomeric state after 180 s. Half-lives of both reactions were estimated at about 10−20 s (Figure 2d). Light irradiation also altered the fluorescence spectrum (Figures 2e and S2a). Importantly, 10 cycles of repetitive photoswitching of the PVA resulted in only a little photobleaching (Figures 2f and S3). These experiments confirmed that the intrastrand photo-crosslinking of PVAs in a single-stranded SNA occurred rapidly, reversibly, and efficiently. Next, we evaluated the effect of an interval between two PV As on the photo-cross-linking reaction by analysis of SNAP2P, SNA-P1P, and SNA-P0P. All three SNAs showed similar reactivity upon irradiation in a single-stranded state (Figures S2 and S4). The cross-linking reactions obeyed first-order reaction kinetics, demonstrating that reactions were intramolecular (Figure S5a, c). A blue shift of the absorption band and a red shift of fluorescent emission of the SNA-PnP series with respect to SNA-P clearly proved formation of PVA-excimer at ground state (Figure S6). We assume that the PVAs are prestacked to form an excimer, which facilitates cross-linking in the single strand. The reverse reaction was slightly suppressed when PVA in SNA-P0P (Figure S5b). Even though the reaction was slightly slower than in the other oligomers, irradiation with 9487

DOI: 10.1021/jacs.9b03267 J. Am. Chem. Soc. 2019, 141, 9485−9489

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was single stranded. As expected, no melting transition was observed after irradiation at 455 nm, proving that the duplex was dissociated (Figure 3b). Upon irradiation with 340 nm light, 90% of the cross-linking was reversed within 15 min (Figure S10b), clearly demonstrating efficient cycloreversion. It should be noted that cycloreversion in the presence of complementary RNA was more effective than in its absence (67% recovery when single stranded). By virtue of the high reactivity of the cycloreversion, irradiation with UV light restored sigmoidal melting (Figure 3b). Irradiation with 455 nm light eliminated the induced CD at around 400 nm, derived from excitonic interaction between the two PVAs before irradiation, and lowered the intensity at 260 nm due to cycloaddition and dissociation of the duplex (Figure 3c). Irradiation with 340 nm light restored the initial CD signals. In the presence of the complementary RNA, repetitive photoregulation by alternating irradiation with 455 and 340 nm light was also possible without significant photobleaching (Figure S10e, f). We constructed energy minimized structures of the duplex SNA-P0P/RNA (Figure 3d) and single-stranded SNAP0P containing cross-linked PVA (Figure 3e). PVA paired with uracil and distortion of the SNA/RNA duplex structure was not observed. In contrast, the local structure around the crosslinked PVAs in the single-stranded SNA-P0P was disordered. This structural change would induce dissociation of the duplex. Cy3-labeled RNA and anthraquinone-labeled SNA containing two PVA residues were also analyzed by fluorescence spectroscopy (Figures 4a and S11). As estimated from the quenching of Cy3 emission by anthraquinone, about 80% of SNA formed a duplex with RNA at 20 °C before irradiation (Figure 4b). After irradiation with 455 nm light at 20 °C, the emission of Cy3 was recovered, and we estimate that over 70% of duplexes were dissociated. Upon irradiation with 340 nm, emission intensity decreased with about 70% of the duplexes reformed.50 Three cycles of photoisomerization were performed without serious deterioration of duplex formation (Figure 4c). Thus, the fluorescence analysis directly confirmed reversible photoregulation of SNA/RNA duplex formation and dissociation at constant temperature. In conclusion, we successfully developed a system in which SNA/RNA duplex formation and dissociation are controlled by utilizing a novel photoreactive nucleobase analogue PVA. Two PVAs in the SNA strand were cross-linked through intrastrand photocycloaddition upon light irradiation with high conversion ratio, selectivity, and reversibility of the reactions. Importantly, effective photocontrol of SNA/RNA duplex formation was achieved at room temperature (20 °C), and this is the first example of a reversible photoswitching of XNA/ RNA duplex formation. This strategy should be applicable to another acyclic XNA such as PNA and L-aTNA for their photoregulation. Photodimerization of PVA has advantages over azobenzene due to the effective regulation with only two modified residues and the high thermal stability of both states (Figure S13); this differs from the azobenzene system in which heat induces cis-to-trans isomerization. The reported approach using PVA makes SNAs candidates for use as photoregulated biological tools and in photon-fueled nanomachines. In particular, photoregulatable anti-miRNA oligonucleotides composed of SNA (SNA-AMO)20 would be achievable in the near future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.9b03267. Experimental procedures, supporting figures and tables, and NMR charts (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hiroyuki Asanuma: 0000-0001-9903-7847 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI grants JP18H03933 (H.A.), JP17K14514 (K.M.), and JSPS A3 Foresight Program. Support by Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) (H.A.) from the Japan Science and Technology Agency (JST) and by the Asahi Glass Foundation (H.A.) are also acknowledged.

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