Photochemical modifications for DNA/RNA oligonucleotides

Light-triggered chemical reactions can provide excellent tools to investigate the fundamental mechanisms important in biology. Light is easily applicable and orthogonal to most cellular events, and its dose and locality can be controlled in tissues and cells. Light-induced conversion of photochemical groups installed on small molecules, proteins, and oligonucleotides can alter their functional states and thus the ensuing biological events. Recently, photochemical control of DNA/RNA structure and function has garnered attention thanks to the rapidly expanding photochemistry used in diverse biological applications. Photoconvertible groups can be incorporated in the backbone, ribose, and nucleobase of an oligonucleotide to undergo various irreversible and reversible light-induced reactions such as cleavage, crosslinking, isomerization, and intramolecular cyclization reactions. In this review, we gather a list of photoconvertible groups used in oligonucleotides and summarize their reaction characteristics, impacts on DNA/RNA thermal stability and structure, as well as their biological applications.


Introduction
Optical control of chemical reactions has recently gained popularity. [1][2][3][4] These controls rely on photoconvertible groups that undergo structural changes upon irradiation by light. [5][6][7][8][9][10] Light can be readily applied to and removed from a reaction, and the wavelength, localization, and intensity of the light can be precisely controlled. 11,12 Thus, light offers distinct advantages in triggering and controlling reactions, compared with other more common methods such as chemical inhibition, rapid mixing, temperature-, salt-or pH-jumps. In some reversible reactions, the forward or reverse reactions are promoted by distinct wavelengths of light, offering a unique advantage. 13,14 Dr Amirrasoul Tavakoli received his Bachelor's degree in Chemistry and Master's degree in Nanotechnology from the University of Tehran (Tehran, IRAN). He obtained his PhD degree in Biochemistry under the supervision of Prof. Jung-Hyun Min from Baylor University (Texas, USA) on light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein using photoreactive DNA. He is currently a postdoctoral fellow in Prof. Daniella Nicastro's lab at the University of Texas Southwestern Medical Center (Texas, USA), developing methods for localizing and identifying specic molecules inside cells at near-atomic resolution using cryo-electron tomography.
Prof. Jung-Hyun Min is an Associate Professor of Chemistry and Biochemistry at Baylor University (Texas, USA). She received her Bachelor's degree in Chemistry from Seoul National University (Seoul, South Korea) and a PhD degree in Biochemistry from University of Washington (Washington, USA). She conducted postdoctoral research in Structural Biology at the Memorial Sloan-Kettering Cancer Center/Howard Hughes Medical Institute in New York, USA. She was also an Assistant/Associate Professor of Chemistry at the University of Illinois at Chicago before moving to Baylor. Her research focuses on determining the mechanism of the nucleotide excision repair process in eukaryotes using various structural, biochemical and biophysical tools including photoreactive oligonucleotides.
Photoconvertible modications in small molecules, 15,16 oligonucleotides, 12,17,18 peptides, 19 and proteins (mostly enzymes) [20][21][22][23][24] have been applied to control and monitor biological events such as gene expression, enzyme activity, oligomerization states, cellular localization, and immune responses. Here, we have compiled a list of photoreactive modications on DNA/RNA oligonucleotides and summarized the literature on their reaction characteristics, impacts on DNA/RNA thermal stability and structure, as well as their biological applications (Table 1).
Many photochemical groups entail 'bulky' modications that alter the DNA/RNA structures in unique ways and some modications can induce site-specic strand-breaks, thus mimicking cellular DNA damage. Thus, these applications may be applicable to studying various DNA damage repair and response mechanisms as well as in the more commonly used applications such as gene expression control. We hope this review will provide useful information for the community of researchers looking for ways to use light to study biochemical/ molecular events.
Photochemical modifications for DNA/ RNA oligonucleotides Photochemical modications are most commonly incorporated into oligonucleotides by solid-phase synthesis using phosphoramidite chemistry in which a phosphoramidite building block containing the desired photochemical group is rst synthesized and subsequently incorporated to an oligonucleotide chain. 25 Post-synthetic approaches have also been used in which site-specic chemical reactions were carried out directly on nucleic acids: this approach can bypass the need for specialized equipment such as DNA/RNA synthesizer. [26][27][28] Most of the modications in this review are incorporated into oligonucleotides via the phosphoramidite chemistry unless noted otherwise.

o-Nitrobenzyl
ortho-Nitrobenzyl (oNB) group is the most extensively studied and applied photoremovable group. A wide variety of functional groups can be introduced into the oNB scaffold, and oNB derivatives have been used as a part of DNA, RNA, small molecules, and proteins. 11 Photocleavage wavelengths are tunable (l ¼ 345-420 nm). 29 In DNA/RNA, they can be incorporated in the backbone, ribose, or nucleobase. Initially, oNB derivatives have been employed as a part of the backbone linkers that can trigger light-induced strand breaks (Fig. 1). [30][31][32] These cleavable oNB linkers have been used in various systems including circular antisense oligonucleotides, 33 DNAzyme, 34 negatively charged peptide nucleic acids, 35 single-stranded circular RNAs as RNA interference (siRNA) precursors, 36 single guide RNA (sgRNA) for CRISPR-Cas9-based gene editing, [37][38][39] and splice-switching oligonucleotides. 40 Caging the 2 0 -OH with oNB group 41,42 has been applied to regulate DNAzymes by the Lu group ( Fig. 1). [43][44][45][46] oNB and its derivatives have also been incorporated in nucleobases to make caged nucleobases for various applications (reviewed by Deiters 30 ). 1-(ortho-Nitrophenyl)-ethyl (NPE) and 2-(ortho-nitrophenyl)-propyl (NPP) caged nucleotides were among the rst oNB-modied nucleobase. 30 Later, 6-nitropiperonyl methyl group (NPM) on N 4 -dC and its corresponding hydroxymethylene analogs (NPOM) on N 3 -dT, N 3 -U and N 1 -dG were developed; these groups offered longer photocleavage wavelengths ($365 nm) than that used for oNB and better stability in an aqueous environment at various pHs. 30 In particular, NPOM, developed by the Deiters group, has been extensively used for various in vitro and in vivo biological applications. 9,40,[47][48][49][50][51] Closely related propargyl-6-nitroveratryloxymethyl (PNVOM) modication contains an alkyne group available for postsynthetic click reaction. 52 The nitrodibenzofuran (NDBF) group attached to N 3 -dT 53 or N 4 -dC and N 6 -dA 54 showed photocleavage with >400 nm wavelength. 54 4,5-Dimethoxy-2-nitrophenylethyl (DMNPE) has been used to modify internal 55 and termini 56 phosphate in siRNA. 1-(4-(2-(Dimethylamino)ethoxy)-5-methoxy-2-nitrophenyl)ethyl carbonyl (DMNEC) moiety has been utilized for acylating 2 0hydroxyls of RNA. 26,57 Reaction characteristics. In oNB and its derivatives, the caged substrate such as oligonucleotide (X in Fig. 1 center) can be attached to the benzylic ring as a leaving group to be released upon irradiation via Norrish type II mechanism, mediated by radicals ( Fig. 2; extensively reviewed in ref. 11). Substitutions on the benzylic ring affect the stability as well as the absorption spectra of the caged molecules. 29 An electronwithdrawing group at the para-position to the nitro group of oNB or a moderately electron-donating group in the metaposition results in a red-shi in the absorption and licenses cleavage with longer wavelengths of light (reviewed in ref. 29).
oNB derivatives in oligonucleotides are shown to be removed in seconds to minutes range using a wide range of power mW-W. 39,49,58,59 In a study by Stephanopoulos et al., 85% removal of NPOM-caged DNA occurred in 3 s using 18.2 W lamp. 60 Thermodynamic or structural characteristics. In a comprehensive DNA duplex melting study by Heckel et al., the oNB derivatives including NPP, NPE, NPOM, and NDBF were shown to generally lower the melting temperature (T m ) of 15-mer DNA duplexes by 6-16 C, which is also affected by sequence. 61 Notably, the T m of NPE groups in DNA duplexes was also sensitive to the conguration of the stereogenic center (indicated as * in Fig. 1): (S)-NPE group decreases T m by 9.2 C versus that of the unmodied sequence, a larger decrease relative to a 4.8 C decrease by the (R)-NPE. 62 NOE-based structural analyses revealed that both enantiomers retained Watson-Crick base pairing of the NPE-modied dC base and its partner, but the different NPE stereoisomers interacted with neighboring bases differently, resulting in the differential impact on its thermal stability. 62 Min et al. showed T m of NPOM-caged 24-mer duplex DNA is $7 C lower than that of the unmodied DNA, while the T m of NPOM-DNA aer photocleavage was the same as that of the unmodied DNA. 58 Molecular dynamics simulations of NPOM-dT containing DNA indicates that NPOM may occupy in the major groove of the DNA as the nucleobase takes up a syn conformation. 58 Introduction of three NPOM groups over 14-bp duplexed region within a DNA hairpin, the melting temperature decreased by $30 C. 9 The impact of NPOM modication in U or G in RNA duplex (21-bp) also depended on the position and number of modications. 47 Heckel et al. also reported that NDBF on N 4 -dC and N 6 -dA in 15-bp DNA duplexes lowered the Fig. 1 Representative structures of oNB modifications on oligonucleotides. oNB's X represents a caged substrate such as an oligonucleotide. Other types of oNB derivatives are also available but not shown for clarity.
T m by 16 C and 12 C compared with the unmodied duplexes, which were larger decreases than those caused by NPE modications in the equivalent positions (DT m ¼ À8 C and À6.2 C). 54 Biological applications. oNB family of modications are the most versatile, and each was used to photo-regulate nucleic acid functions in various ways.
NPP and NPE modications. NPP-dT and NPE-dT were used to block the binding of the MutS mismatch repair protein to a DNA bulge, which then could be removed by photoirradiation and enable the binding. 63 NPP, NPE, and oNB linkers were also widely used as photocleavable linkers in the backbone of oligonucleotides to control gene expression and editing. For instance, NPE has been utilized in modulating siRNA activity. 64 In a recent work, singlestranded RNA circularized via an oNB linker in the phosphodiester backbone was used as siRNA precursor, which could efficiently be activated to linear RNAs by 365 nm irradiation in vitro. 36 Various types of oNB-based linkers were also used: e.g., as internal photocleavable linkers within single guide RNA (sgRNA) to inactivate Cas9 nuclease and attenuate genome editing by CRISPR-Cas9 within cells 37,39 and as a way to control RNA-cleaving DNAzyme's activity. 34 NPOM. The ability of NPOM to disrupt DNA and RNA hybridization has been used in various applications such as DNA nano-tweezer, 60 DNA triplex nanostructures, 65 DNA computation, 66 as well as controlling antisense DNA agent activity, 52 DNAzyme activity, 50,67 restriction endonuclease, 49 and polymerase chain reaction. 68,69 NPOM was also used as a part of gene promoters, triplex-forming oligonucleotides, microRNA, siRNA 47 as a tool to regulate gene transcription and translation. In more recent works, NPOM was applied to the CRISPR-Cas9 gene editing system. In one study, NPOM-caged guide RNAs (gRNAs) conferred complete suppression of gRNA:dsDNA-target hybridization, which could subsequently be restored with light irradiation. 51 In another study, NPOM-modied gRNA hybridized with DNA and allowed Cas9 to bind DNA, but the gene cleavage was suppressed until light-induced activation. 70 This approach, referred to as very fast CRISPR (vfCRISPR), also created double-strand breaks (DSBs) at a submicrometer scale within seconds, which could be used to track the recruitment of DSB repair proteins to the damaged sites. 70 Min et al. also showed that NPOM-modied dT could be specically bound by the Rad4/XPC DNA nucleotide excision repair protein and that such binding was abolished upon light-induced photocleavage of NPOM. 58 Other biological applications of NPOM include modulating mRNA splicing (splice switching) in cells and zebrash 40 and controlling TLR9 in immune responses. 18 DMNPE & DMNEC. DMNPE was used to control the activity of siRNA as a part of the internal backbone or its termini. 56 For instance, the regioselective incorporation of DMNPE groups in the four phosphate termini of an siRNA duplex effectively limited the RNAi activity, which could be restored upon irradiation. 56 On the other hand, the DMNEC modications were used to post-synthetically acylate 2 0 -hydroxyls of RNA ribose. Hammerhead ribozyme activity could be photo-regulated using this method in which multiple DMNEC groups were incorporated along the RNA molecule. 26 Later, Zhou et al. used DMNEC on gRNA to suppress CRISPR-Cas gene editing, which could be reversed by 365 nm light. 57 2. p-Hydroxyphenacyl p-Hydroxyphenacyl (pHP) modication has been rst introduced by the Reese group. 71 Currently, pHP modication and their photolysis reactions have been reported for base modications on N 3 -dT, 59 O 4 -dT, 72 and O 6 -dG (Fig. 3). 73 Addition of a benzothiazole to pHP as 2-(2 0 -hydroxyphenyl)benzothiazole (HBT) has also been introduced as fast decaging moiety on O 6 -dG (Fig. 3B). 74 Reaction characteristics. pHP photosolvolysis typically occurs far more rapidly following excitation compared with the more commonly used oNB derivatives (Section 1), which proceeds through an intermediate that can exist for seconds to a minute. The deprotection rate of pHP correlates inversely with the pK a of the conjugate acid of the leaving group. The absorption spectrum also changes drastically as the reaction progresses from a conjugated phenyl ketone to a nonconjugated phenol, 4-hydroxyphenyl acetate. 11 The photocleavage reaction of pHP on N 3 -dT was slow (1 h using 313 nm), 59 but the incorporation of pHP in O 4 -dT shortened the photodecaging, and complete photodecaging was achieved in 0.3 min. 72 pHP at the O 6 -dG position was decaged with a time constant t 1/2 of 17 s upon irradiation with 295 nm UV light. 73 Later, Singh et al. 75 improved the photodecaging reaction by synthesizing HBT. HBT has strong uorescence and Fig. 2 oNB photocleavage reaction. Upon irradiation with UV-A light, the bond between the oNB and the leaving group (e.g., dC nucleoside) is cleaved in a radical-mediated reaction. The byproduct (e.g., CO 2 ) varies depending on the type of oNB photocage.
can use longer wavelength light (400 nm) for decaging. HBT-modication on O 6 -dG cleaved using blue light (405 nm) in a short pulse (#30 ms) was used for rapidly initiating the folding and activation of twister ribozyme in a single molecule study. 74 Thermodynamic or structural characteristics. pHP modications are reported to be thermodynamically destabilizing for DNA duplexes. pHP-caged N 3 -dT or O 4 -dT can destabilize 15bp duplexes DNA by $9 C. 59,72 pHP-caged O 6 -dG is also proposed to prevent RNA annealing due to the steric hindrance and changes in the base-pair hydrogen-bonding patterns. 73 Biological applications. pHP modications were shown to temporarily block the antisense pairing between non-coding RNAs catalyzed by the RNA chaperone Hfq 62 and to regulate the function of the twister ribozyme. 73,74 The fast uncaging of pHP and its derivatives could be promising for various timeresolved studies that require the photoremoval reaction to occur faster than the molecular process under investigation. 73 3. TEEP-OH (thioether-enol phosphate, phenol substituted) Photolabile TEEP-OH (thioether-enol phosphate, phenol substituted) was inspired by the p-hydroxyphenacyl bromide group (pHP, Section 2) 76 and can be incorporated via postsynthetic modication on the phosphodiester backbone of phosphorothioate DNA. 28 Upon light irradiation, TEEP-OH is photocleaved and the phosphate backbone reverts to its native form (Fig. 4). This modication was used for photoregulation of an RNA-cleaving DNAzyme, a G-quadruplex peroxidasemimicking DNAzyme, and a thrombin-binding aptamer. 27,28 Reaction characteristics. The photodecaging of the RNAcleaving DNAzyme was carried out using 365 nm light (12 W hand-held UV lamp) for 15 minutes. 28 In a different application with G-quadruplex, the photoreaction was carried out by  300 nm light (12 W hand-held UV lamp) with the sample-tolamp distance of 5 cm for 20 minutes. 27 Thermodynamic or structural characteristics. Not reported. Biological applications. TEEP-OH modication of RNAcleaving DNAzymes in their active sites signicantly inhibited the DNAzyme's activity. Upon light irradiation at 365 nm, the activities were restored to those of the native enzyme. The photodecaging and restoration of DNAzyme activity could also be accomplished when the DNAzyme and its substrates were transfected into HeLa cells. 28 TEEP-OH modication of a Gquadruplex DNAzyme also inhibited the DNAyzme's peroxidase activity, which could be restored by UV photocleavage. 27 TEEP-OH photocaging also could inhibit the activity of thrombin-binding G-quadruplex aptamer, which could be restored upon decaging by UV light. 27

Aryl sulde
Originally reported by the Greenberg group as a way to study nucleobase radical formation, electron-rich aryl sulde (ArS, dimethoxythiophenyl) undergoes carbon-sulfur bond homolysis upon irradiation. 77 ArS-modied nucleobase (e.g., on C 5methyluridine or C 6 -hydrothymidine) disrupts nucleic acid structure by perturbing base stacking. 77,78 ArS on 5-methyluridine prevents RNA hairpin formation in short RNA as well as the folding of the preQ1 class I riboswitch. 78,79 Reaction characteristics. Photolysis produces a native nucleotide via a radical pair that undergoes disproportionation within a solvent cage upon irradiation with light at 350 nm ( Fig. 5). 78 The reaction was complete within minutes and the rate is estimated to be very fast, in the order of microseconds, based on thiol competition experiments. 78 Thermodynamic or structural characteristics. ArS is shown to disturb the base-pairing and thus secondary structure of the A-form RNA hairpin, as monitored by CD spectrometry. 78 ArS modication also thermodynamically destabilizes DNA and RNA duplexes. 77,78 For instance, ArS-modied hydrothymidine in a 12-bp DNA duplex lowered the T m by 10 C. 77 Biological applications. In studies by Greenberg et al., the incorporation of ArS inhibited the folding of a preQ1 class I riboswitch that binds to the preQ1 ligand to form RNA pseudoknot. 78 Such inhibition of RNA folding could be abolished upon the photocleavage of ArS. 78

Nitroindole group
Photocleavable nitroindole nucleoside was introduced by Lhomme and colleagues. 80,81 Irradiation (l ¼ 350 nm) of oligonucleotides containing 7-nitroindole or 5-nitroindole triggers a radical process: the excited nitro group induces an intramolecular H1 0 abstraction leading to the release of a nitrosoindole group while forming a highly labile deoxyribonolactone, an abasic site (Fig. 6). 82 Subsequent mild basic or thermal treatment leads to cleavage of the DNA backbone via band d-elimination at the abasic site. 82 Reaction characteristics. The photocleavage reactions of nitroindole-modied DNA nucleoside accompany changes in the absorption spectra. Two isobestic points at 310 nm and 365 nm were observed in the UV spectra of the irradiated solution of the free nucleoside, which is monitored and characterized by nitrosoindole (l max ¼ 406 nm) and deoxyribonolactone (l max ¼ 241 nm) formation in different time intervals. Photolysis of 7-nitroindole-containing oligonucleotides with 350 nm UV-A light was completed in a few minutes (t 1/2 ¼ 1.0 min). 83  Thermodynamic or structural characteristics. 7-Ntroindole and 5-nitroindole DNA nucleosides have both been shown to lower the T m of DNA duplexes. 7-Nitroindole was slightly more destabilizing than 5-nitroindole: 82 13-15 C lower T m for 7nitroindole than that for unmodied DNA versus 10-11 C lower T m for 5-nitroindole in the same 11-bp DNA duplex context. 82 Biological applications. Light-induced photocleavage of nitroindole was used to induce controlled release of DNAbinding proteins such as NF-kB as a part of the catch-andrelease DNA decoys where 7-nitroindole could be used in place of regular dG's in the NF-kB binding sequence. 83

Benzophenone and acetophenone
Benzophenones contain a carbonyl carbon that undergoes intersystem crossing in high yields, making it a robust triplet photosensitizer for use in organic and biological chemistry. 84 While a wide range of applications have utilized its lightinduced C-C photocrosslinking properties, 85 the Rentmeister group reported that benzophenone modication at N 7 -G in the context of RNA oligonucleotides can undergo a photocleavage reaction (Fig. 7). 86 Upon irradiation with 365 nm light, benzophenone is cleaved from nucleobase through hydrogen abstraction mechanism. The photocaged guanosines used as a 5 0 -cap blocked the RNA's interactions with the translation initiation factor eIF4E and the RNA decapping enzyme DcpS. 86 Benzophenones and acetophenones also can form UV-induced (C-C) cross-links with protein amino acids. 84 For example, terminal deoxynucleotidyl transferase enzyme could be crosslinked to 3 0 -tails of DNA containing benzophenones and acetophenones on N 4 -dC's using 365 nm light ( Fig. 8) 87 Reaction characteristics. UV irradiation (365 nm) of benzophenones generates C-O biradical through n-p* transition, which can lead either to photocrosslinking or its reversal, photocleavage. 84,85 The photocleavage reaction was rendered complete aer 10 min of irradiation at 365 nm. 86 Thermodynamic or structural characteristics. None reported.
Biological applications. Benzophenone modication of an N 7 -G blocked the interaction between the 5 0 cap in the mRNA and the translation initiation factor eIF4E and the mRNAdecapping enzyme DcpS. Photocleavage followed by remethylation of the N 7 -G in 5 0 cap (GpppA to m 7 GpppA) restored the binding with these proteins. 86 On the other hand, benzophenone-and acetophenone-modications on N 4 -dC could generate protein-DNA crosslinks with the bound terminal deoxynucleotidyl transferases using 365 nm light. 87

Coumarin
Coumarin-based groups are widely used photo-removable groups because of their large molar absorption coefficients at longer wavelengths, high release rates, and uorescent properties. They are also capable of photo-crosslinking via [2 + 2] cycloaddition. One of the representative coumarin derivatives, (7-diethylaminocoumarin-4-yl)methyl (DEACM) was rst introduced by Hagen et al. 88 (Fig. 9). DEACM could be introduced in the backbone of the DNA or on N 3 -dT, 72 O 6 -dG, 89 and O 4 -dT (without and with a linker as in DEACM-O-Bn-dT) 90,91 as well as on the g-phosphate group of ATP (Fig. 9A). 92 6-bromo-7hydroxycoumarin-4-ylmethyl (Bhc) was rst introduced by Tsien et al. 93 Bhc has been employed in modifying C 4 -dC (as Bhcmoc or Bmcmoc), 94 5 0 -position of the ribose in adenosine, 95 and the phosphate backbone (Fig. 9B). 96,97 The mechanism for photo-removal of these coumarin derivatives is through solventassisted photo-heterolysis (S N 1 mechanism). Coumarin-modied oligonucleotides have been applied in various ways, for instance, in generating DNA strand breaks (e.g., by using DEACM linker in Fig. 9A) and in regulating DNA polymerization, translation and transcription. Inspired by Bhc, a series of photo-removable groups based on quinoline was reported by Guo et al. Among them, 8-bromo-2-diazomethyl-7hydroxyquinolinyl (BHQ-diazo) showed the highest caging and restoration efficiency for the anti-thrombin aptamer HD1 (Fig. 9C). 98 Reaction characteristics. The photocleavage reactions of coumarin derivatives can be accomplished by a broad range of light wavelengths (350-470 nm) (Fig. 10). 6,94,95,99 Reported reaction times range from seconds to minutes. 59,72,89,94,95 The presence of the extended spacer in DEACM-O-Bn-dT leads to fast decay and no byproduct. 90 DEACM-containing oligonucleotides exhibit a very intensive red-shied absorption band (l max ¼ 398 nm) compared with oNB (l max ¼ 365 nm), from the p-p* transitions of the coumarin chromophore. 89 (S)-diphenylmethyltriazole-coumarin (DPMTC) O 4 -dT derived from postsynthetic Cu-catalyzed azide-alkyne on DNA (i.e., click chemistry) could be uncaged with 405 nm light within minutes. 95,103 For the quinoline-derivative BHQ, photolysis occurs through solvent-assisted photoheterolysis (S N 1) reaction mechanism as with the coumarin family. 100 Light irradiation at 365 AE 5 nm with an approximate dose of 100 mJ cm À2 , caged anti-thrombin aptamer HD1 was released more than 90% within 30 s. 98 In addition to the photocleavage reaction, coumarin molecules can also undergo reversible photocrosslinking via a photoinduced [2 + 2] cycloaddition reaction similarly as psoralen (Fig. 11). 101,102 The photocyclization reaction between coumarin and thymidine leads to fast and quantitative DNA interstrand crosslink (ICL) formation (>98%). 101 The DNA crosslinks were generated by 350 nm irradiation whereas the reverse reaction, cyclo-reversion of the photo-adducts were achieved by 254 nm light (Fig. 11). 101 ICL formation between a coumarin moiety containing a exible two-carbon or longer chain and thymidine on the opposite strand completely quenches the uorescence of coumarin, which allows for the monitoring of DNA crosslinking process over time via uorescence spectroscopy. 101 DNA crosslinking by coumarins shows a kinetic preference when anked by an A:T base pair as opposed to a G:C pair. 101 Thermodynamic or structural characteristics. In a comprehensive DNA duplex melting study by Heckel et al., the coumarin derivatives (without crosslinking) were shown to generally destabilize the DNA duplex. 103 89 Biological applications. DEACM-incorporated photocleavable linker was used to catch and release NF-kB, a DNAbinding transcription factor, whereby photocleavage (365 nm) and subsequent DNA strand break abrogated the binding. 99 DEACM-ATP used as photocaged ATP: its uncaging via remote light (400 nm) was used for transient DNA polymerization. 92 DEACBY alkyne was used to inhibit duplex formation between a circular DNA and its target, which could be restored upon light irradiation. 91 Bhc-caged mRNA has been used to control the translation activity of mRNA in vitro and in vivo. Illumination with 350-365 nm ultraviolet light removed Bhc from caged mRNA, resulting in a recovery of translational activity. 96 Also, BHQ-diazo as a modication on the phosphate backbone group of a 15-bp anti-thrombin aptamer HD1 inhibited the thrombin binding, which could be restored upon light irradiation. 98 Applications of coumarin-based DNA crosslinking in a biological context remains to be seen.

Carbazole
The photo-crosslinking has been widely used to stabilize complexes with DNA by a covalent-bond formation. 104,105 For instance, photo-crosslinkers such as psoralen (a member of furocoumarin family, related to coumarin, Fig. 11) can produce DNA inter-strand crosslinks via [2 + 2] cycloaddition reaction when irradiated by UV-A (365 nm) either via their furan or pyrone photoreactive site. The crosslinks can be reversed upon irradiation 254 nm shorter wavelength. Although psoralen is widely used as a thymine-selective photo-crosslinker in biological studies, there are limitations such as requiring a TpA step in the sequence and causing photodamage to DNA by forming pyrimidine photodimers upon cycloreversion that uses UV-C (254 nm). 104,106 To alleviate these issues, Fujimoto et al., reported 3-cyanovinylcarbazole nucleoside ( CNV K) as a reversible photo-crosslinker that can photo-crosslink to pyrimidine base located 5 0 to the complementary base through [2 + 2] cycloaddition with 385 or 365 nm irradiation (Fig. 12). 106 The resulting photo-adducts can be uncrosslinked by 312 nm irradiation without causing DNA damage. 106 CNV K shows higher reactivity compared with psoralens, showing 97% yield with 1 s of longer wavelength UV-A light (366 nm). CNV K nucleoside was further developed to improve photoreactivity. 3-Cyanovinylcarbazole-modied D-threoninol ( CNV D) which has a exible structure showed enhanced photoreactivity for the pyrimidine base at the À1 position in the complementary strand (Fig. 13): the photoreactivity of CNV D was 1.8-(for crosslinking with dT), 8-(with dC), and 2.8-fold (with U in RNA) greater than that of CNV K. 107 PC X, pyranocarbazole nucleoside was developed to use visible light instead of UV-A, therefore less toxic and damaging (Fig. 13). 14 Recently D-threoninol version of the PC X photocrosslinker ( PCX D) was reported, showing a higher photoreactivity than PC X (Fig. 13). 8 In addition, n-CNV K with variable linker lengths (n ¼ 2-5) was developed to use with click chemistry. 108 This probe is capable of photo-crosslinking with pyrimidine bases at locations other than the À1 position (Fig. 13). 108 Reaction characteristics. CNV K, CNV D, PC X, and PCX D can photo-crosslink to pyrimidine bases within a few seconds of photoirradiation. NMR, kinetic, and structural analysis indicated that the photo-crosslinking reaction with thymine proceeds with trans isomer of CNV K, and one single photo-adduct. However, these photo-crosslinkers can only crosslink to the counter base if it is adjacent to the 5 0 -side (À1) position to the crosslinker-containing base (5 0 -nXn-3 0 and 5 0 -Ynn-3 0 where X is the crosslinker-containing base and Y indicates the position of the crosslinked pyrimidine). 8,14,[106][107][108][109][110][111][112] Fujimoto et al. reported that the rate constant of the photocrosslinking reaction of CNV D is 0.106 s À1 which is comparable to CNV K (0.059 s À1 ). Within the same sequence, psoralen showed a much slower photo-crosslinking rate (0.003 s À1 ) using the same wavelength (365 nm). 110 The reaction rate constant of PCX D with cytosine is 4.3-fold larger than that of PC X. 8 Thermodynamic or structural characteristics. Molecular modeling studies indicated that photochemical [2 + 2] cycloaddition is facilitated with the orientation of the vinyl group of CNV K to be stacked onto the C5-C6 double bond of pyrimidine nucleobases located at the À1 position on the complementary strand (see above). 104,111 Overall, crosslinking stabilizes the oligonucleotide duplexes. 14,108,111 The impacts of the photoconvertible groups on duplex stabilities before crosslinking reaction were also investigated by the Fujimoto group. In general, a exible threoninol linker (e.g., CNV D) destabilizes the duplex than having a regular phosphodiester linker with deoxyribose (e.g., CNV K): in a 9-bp DNA duplex, CNV D was more destabilizing than CNV K by 5 C. 8 Also, pyranocarbazole (e.g., PC X or PCX D) is more destabilizing than 3-cyanovinylcarbazole (e.g., CNV K and CNV D). PCX D-containing duplex has the lowest melting compared to the others. 8 Biological applications. Vinylcarbazole-based photocrosslinkers have been used for applications such as targeted site plasmid labeling, 113 transient transgene silencing, 114 and identifying targets of endogenous small RNAs. 115 CNV K and PC X have been used for detecting locations of RNA, 7,116 and methylcytosines in DNA in cells by uorescence in situ hybridization (FISH). Incorporating multiple crosslinkers could help increase the sensitivity of FISH by 40-fold in the region where detection was difficult due to complex secondary structures using conventional FISH. 112 Fujimoto group reported the use of CNV K photocrosslinking in antisense DNA technology: photocrosslinkable antisense oligonucleotides containing CNV K can regulate GFP expression in a sequence-specic manner only aer 10 s photocrosslinking with 365 nm light in HeLa cells. In a recent study, they investigated and compared the photo-crosslinking rate and its inhibitory effect including CNV D, CNV K, and psoralen on gene expression. 110 The inhibitory effect on gene expression was the highest with CNV D (93%), while no inhibitory effects were observed with psoralen.
In another recent study, they regulated the DNAzyme activity by photoirradiation through the photochemically reversible formation of covalent bonds. 109 While photo-crosslinking using 365 nm completely abolished the activity of the DNAzyme harboring CNV K, uncrosslinking using 312 nm irradiation   restored the activity. 109 C NVK also was shown to accelerate in vitro DNA strand displacement reactions, which may be employed for rapid-response DNA nano device technology using higher-order DNA structures. 117

Vinyl derivatives
As with the vinyl groups in 3-cyanovinylcarbazole (CNV) modi-cations (Section 8), other DNA/RNA modications containing vinyl group have been reported with photo-crosslinking properties (Fig. 14A). In addition, some vinyl-derivatives can also undergo cis-to trans-photoisomerization around the C-C double bond in the absence of a crosslinkable partner; this then alters the orientations of the attached moieties and subsequently the DNA/RNA structures (Fig. 15A). 118 Here, we discuss the vinyl derivatives reported for these two reaction categories.
9A. Vinyl derivatives for photocrosslinking via [2 + 2] cycloaddition. p-Stilbazole photo-dimerization was rst illustrated by Asanuma et al. 119 p-Stilbazole positioned opposite of each other in DNA duplex, linked through D-threoninol linkers could be crosslinked with UV light, thus signicantly stabilizing the duplex (Fig. 14B). NMR analyses indicated that two diastereomers are produced on photo-crosslinking due to rotation of vinyl group. 119 Later, a stilbene derivative, p-cyanostilbene, was introduced at the termini of siRNA in both strands and photocrosslinking resulted in "termini-free" siRNAs which could not be cleaved by dicer that requires 3 0 overhang ends from the precursor siRNA (Fig. 14B). 120 Similarly, styrylpyrene (Sp) pairs introduced in complementary positions in DNA duplexes could undergo a [2 + 2] cycloaddition photocrosslinking reaction by visible light irradiation (l ¼ 455 nm) whereas UV light (l ¼ 340 nm) could reverse the crosslinks (Fig. 14B). 121 More recently, 8-pyrenylvinyl adenine ( PV A) was employed as a way to control duplexation between serinol nucleic acid (SNA) and RNA (Fig. 14C). When incorporated in SNA in adjacent positions, PV A could undergo intrastrand photodimerization by 455 nm light, which abolished the duplexation with a complementary RNA. However, the crosslinks could be reversed with cycloreversion catalyzed by 340 nm light. 122 It is noteworthy that both the forward and reverse reactions could be carried out to completion at constant room temperature. 122 8-Naphthylvinyladenine ( NV A) is also used in SNA for crosslinking/ uncrosslinking reaction, similarly as PV A, but uses a shorter wavelength of light than PV A: intrastrand crosslink by irradiation with 340-405 nm light and reverse reaction by #300 nm light. In an SNA strand with adjacent NV A and PV A residues, irradiation with 405-465 nm led to intrastrand crosslink, which was reversed by irradiation with #340 nm light. 123 In all these cases, the intrastrand photo-crosslinking destabilize SNA/RNA duplexes, resulting in duplex dissociation while its cycloreversion led to duplex formation. 123 With these NV A/ NV A and NV A/ PV A photo-switches, the hybridization states of SNA/RNA duplexes could be independently controlled by using light of varying wavelengths. 123 Reaction characteristics. Strylpyrene pairs (Spa and Spb) introduced as a part of D-threoninol linker in the opposite strands of DNA duplex could also undergo [2 + 2] photocycloaddition using visible light (l z 455 nm), but gave two diastereomers as a result of the rotation of the styrylpyrene residues. 121 The reaction progress and stacking of the Sp dimers could be monitored using UV-Vis absorption spectroscopy. Upon visible light irradiation of S P a/S P b at l ¼ 455 nm, the absorption band at l z 390 nm decreased with irradiation time and almost disappeared aer 60 min of irradiation while new bands concurrently appeared at l ¼ 338 and 354 nm. The progress of photocycloaddition was apparent also due to changes in color and uorescence of the solutions from colored to colorless. 121 PV A-containing oligonucleotide features an absorption band at around 400 nm (characteristic of vinylpyrene) and upon irradiation with 455 nm light immediately decreased and almost disappeared aer 2 min using 203 mW cm À2 power. 122 Simultaneously, new bands appeared at 270 and 354 nm, which correspond to absorption bands of alkylpyrene, a product of crosslinking. Upon irradiation of the crosslinked product with 340 nm light, the initial absorption bands were restored, indicating the recovery of PV A monomers. 122 The crosslinking and uncrosslinking reactions were rendered complete aer 1 h (irradiation at 455 nm) and 15 min (340 nm), respectively. 122 In the case of NV A, irradiation with 405 nm light for 60 s led to the disappearance of the absorption band around 360 nm, and irradiation with 300 nm light of this photo-adduct led to the cycloreversion: 61% of the initial absorption band was recovered within 120 s. 123 Four hybridization states of two SNA/RNA duplexes containing either the NV A/ NV A pair or NV A/ PV A could be orthogonally controlled using different wavelengths of light. 123 Thermodynamic or structural characteristics. Photocrosslinking between Sp groups is thermodynamically stabilizing for DNA duplexes, as expected. Melting measurements revealed that both diastereomer products aer crosslinking had melting temperatures signicantly higher (22-25 C) than that of the uncrosslinked dimer, S P a/S P b. Melting measurements also indicated that the crosslink had been reversed upon cycloreversion. 121 PV A slightly destabilized the duplexes when compared with the unmodied SNA/RNA duplex. 122 On the other hand, NV A in SNA/RNA duplex slightly increased T m compared with the control SNA/RNA 123 while NV A intrastrand photo-crosslink caused severe destabilization of a SNA/RNA duplex containing NV A or PV A: this resulted in the melting of the duplex to single strands. The reverse reaction, cycloreversion, led to the restoration of the duplexes. 123 Biological applications. NV A and PV A were studied as a part of SNA/RNA as mentioned above as reversible crosslinkers that stabilize the SNA/RNA duplexes. 121,123,124 9B. Vinyl derivatives undergoing cis-trans photoisomerization. Several vinyl-containing modications have been developed to modulate DNA and RNA oligonucleotides by reversible cis-trans photoisomerization (Fig. 15A). Maeda et al. synthesized three C 8 -substituted 2 0 -deoxyguanosine (dG) with vinyl-containing modications to modulate DNA hybridization by reversible cis-trans photoisomerization: 8-styryl (8ST), 8naphthylvinyl (8NV), and 8-uorenylvinyl (8FV) (Fig. 15B). 125 Rapid and efficient light-induced trans-to-cis isomerization led to changes (1.4-8 C) in the thermal stability of the duplexes even at room temperature. 125 These nucleosides in the trans forms have little inuence on the B-form structure when duplexed, and their intrinsic uorescence can be used to monitor the isomeric states since the uorescence intensity dramatically changes upon cis-trans isomerization. For instance, the uorescence emission maximum at 450 nm for trans-8ST G is 6 times higher than cis-8ST G. 125 8FV G was used for reversible photo-regulation of G-quadruplex aptamers to bind with thrombin through cis-trans photoisomerization. 126 Later, the same modications were attached to 5 0 -cap methylguanosine (methylG) of mRNA: 8ST-cap, 8NV-cap, and 8FV-cap were developed (Fig. 15B). 118 8NV-cap and 8ST-cap were used to reversibly regulate gene expressions. 118,127 Reaction & thermodynamic characteristics. In 12-bp duplexes containing 8ST G, 8NV G, or 8FV G, the trans forms of the dG modications were photoisomerized to the corresponding cis forms when irradiated for 5 min with 370, 410, and 420 nm light with 86%, 63%, and 77% conversion efficiencies. In addition, subsequent irradiation for 2 min at 254, 290, and 310 nm yielded the trans forms with 94%, 87%, and 77% conversion efficiencies, respectively. 125 Thermal stability study of 12-bp duplexes containing 8ST G, 8NV G, or 8FV G showed both the cis and trans isomers were thermally stable. 8ST G-containing duplex showed the T m of the trans form was 7.9 C higher than that of the cis form. This is probably due to a difference in the steric hindrance of the benzene ring with its neighboring nucleobase and backbone. In contrast, the T m of the trans forms of 8NV G-and 8FV G-containing duplexes were only 1.6 and 1.4 C lower than the cis forms, respectively. 125 This may indicate that the bulky substituents, naphthalene and uorene in 8NV G and 8FV G, may cause serious steric hindrance with the backbone, even in the trans form. 125 Biological applications. The trans to cis isomerization of vinyl derivatives can regulate oligonucleotide duplex hybridizations 125 and has been applied in various biological applications. 101,127 For example, The mRNA containing the 8NV-cap at the 5 0 -end could be switched between a translating (ON) state when in cis form and a non-translating (OFF) state when in the trans form in a reversible fashion by alternately irradiating with 410 nm or 310 nm light. 118 In addition, 8ST-cap can reversibly regulate translation by controlling the interaction with eukaryotic translation initiation factor eIF4E through its cis-trans photoisomerization in living mammalian cells as shown in PC12 neuronal cell line through its neurite expansion and contraction. 127 Furthermore, trans (E)-to-cis (Z) photoisomerization of the 8ST G was utilized by Zhou et al. to reversibly switch between a B-form and Z-form DNA by alternately illuminating with monochromatic 254 nm and 365 nm light. 128

Azobenzene
Azobenzenes (AzoB) are the most widely used reversible photoswitches in oligonucleotides due to their high quantum yields, fast switching, low rate of photobleaching, easy synthesis, high fatigue resistance (high repeatability of photoswitching), and good thermal stability. 12 Irradiation with UV light converts planar trans-N]N bond with zero dipole to non-planar cis isomer (dipole moment of $3 D), which can be accelerated by heat (Fig. 16). 129 The reverse cis to trans isomerization can be achieved by visible light. 129 AzoB was rst introduced in nucleic acids as a part of a exible backbone linker based on propionic acid by Asanuma et al. 130 and was used to regulate duplex 131 and triplex 5,132 DNA formation.
To enatioselectively introduce AzoB into DNA or RNA, optically pure D-threoninol and L-threoninol linkers were employed. D-Threoninol-linked AzoB (D-tAzo) 133 (Fig. 17A) induces larger changes in T m between the trans and cis isomers than L- Fig. 16 Schematic of reversible azobenzene trans-cis photoisomerization with different wavelengths. This conversion occurs through reversible rotation of planar trans N]N to non-planar cis isomer. l 1 is often in the UV-A range, and l 2 in the visible range. threoninol (L-tAzo) (Fig. 17B) and is now commercially available, making it one of the most commonly used forms of AzoB in DNA/RNA. 12 Introducing methylthio-modication at para-position of azobenzene induced a bathochromic (red-) shi of absorption maximum, allowing trans-to-cis isomerization by 400 nm visible light (Fig. 17C). Additional methylation at the ortho-position of the distal benzene ring enhanced the stacking interaction of trans-azobenzene while further destabilizing cis-AzoB (Fig. 17C). This in turn raised the T m of the trans-form and lowered the T m of the cis-form, and the resulting large DT m enhanced photoregulatory efficiency. More recently, an AzoB modied with a highly branched secondary alkylthio group was incorporated into DNA via an L-threoninol scaffold for which the photoisomerization was carried out by visible light (l ¼ 400 nm for the trans-to-cis reaction with 58% efficiency and l ¼ 520 nm for the reverse reaction) (Fig. 17C). In contrast to other AzoB, these modications also showed that the trans-form is duplex-destabilizing than the cis-form (Fig. 17C). 13 More recently, several modications have been made to improve the modest photoisomerization efficiency of D-tAzo (e.g., 30% of cis-isomer at 37 C with irradiation with 365 nm (ref. 134)) or L-tAzo. 13 For instance, Liang et al., introduced AzoB through R-glycerol linker (R-gAzo) which has improved photoisomerization efficiency to 70-80% at room temperature (Fig.  17D). 135 Later, Asanuma, Heckel, and co-workers developed p-Azo and m-Azo C-nucleosides photoswitches which exhibited complete photoisomerization at room temperature ( Fig. 17E and F). 136 The AzoB group was also incorporated as a part of the backbone in DNA/RNA: Tang et al. introduced 4 0bis(hydroxymethyl)-azobenzene to dumbbell hairpin antisense strand complementary to target RNA at the loop position to reversibly control the stability of the hairpin structure via UV or visible light (Fig. 17G). 137 Desaulniers et al., developed photo-regulatable siRNAs with internal azobenzene derivative spacers (siRNAzos) (Fig.  17H). [138][139][140] A related, tetra ortho-chlorinated azobenzenecontaining siRNAs (Cl-siRNAzos) shied the trans to cis conversion wavelength to 660 nm (red-shi) and was applied in cell culture gene inactivation studies (Fig. 17H). 141 AzoB can also be introduced as a part of the purine ring as 2phenyldiazenyl-substituted 2 0 -deoxyadenosine (dA Azo ) and 2 0deoxyguanosine (dG Azo ) ( Fig. 17I and J). 10 G Azo has been developed by Ogasawara and used as a photoresponsive 5 0 -cap of mRNA in vivo to control protein expression. 142 Reaction characteristics. The UV-vis spectrum of unsubstituted trans-azobenzene shows two absorption maxima: a strong one around 320 nm resulting from the symmetryallowed p-p* transition and a weaker one around 430 nm indicative of the symmetry forbidden n-p* transition. The absorption at about 320 nm leads to rotation around the N]N bond and the formation of the cis isomer. The transition associated with the absorption at 430 nm is related to the cis to trans isomerization. These properties can be inuenced by the substitution of the azobenzene core structure and the choice of solvent.
Thermodynamic or structural characteristics. AzoB derivatives in oligonucleotides are shown to be isomerized in seconds to minutes range using mW range of power. 131,133,136,143 Effect of azobenzenes on duplex stability is reviewed by Feringa. 12 In general, the intercalation of planar trans-AzoB stabilizes the DNA or RNA duplexes whereas cis-AzoB destabilizes due to nonplanarity caused by steric hindrance (Fig. 16). The destabilization effect of the cis-Azo was observed by various research groups and for several different azobenzene nucleoside surrogates. 5,13,131,133,136 In general, the T m differences between the cisand trans-forms of AzoB modications in DNA duplexes are $1-5 C. 130,131,136 dA Azo and dG Azo decreased the T m of 10-bp DNA duplexes by 10-13 C compared with 16 C of m-Azo. 10 The impact of AzoB on the thermal stability of the DNA also depends on the stereochemical environment of the group. For instance, the trans-form of D-tAzoB is more stable than that of L-tAzoB because D-threoninol prefers a clockwise winding, as does the DNA double helix. Cis-form is also more destabilized in D-tAzoB than in L-tAzoB, resulting in a larger trans-to-cis stability difference (DT m ) for D-tAzoB.
Biological applications. As extensively reviewed by Feringa 12,144 and Zhang, 145 AzoB groups have been used in numerous biological applications: regulating hybridization in nucleic acids, 124,146 transcription of T7 RNA polymerase, 143 antisense DNA-mediated gene expression, 147 RNA digestion by RNase H using modied DNA, 148 nano-tweezer regulation, 149 and inhibiting DNA aptamer with thrombin. 150 Newer applications include siRNAzos 151 in gene silencing in cells and in vivo: siRNAzos use AzoB as internal spacers within the sense strand in HeLa cells. 138 siRNAzos in the cis form would distort the siRNA helix, thus rendering it non-functional, but irradiation with UV light would make it functional and lead to gene silencing. 138 siRNAzos also has been used in the 3 0 -end of the sense strand with improved nuclease resistance for gene silencing applications. 139 Red-shied Cl-siRNAzos were used in cell culture with reversibility. 141 Additionally, dG Azo developed by Ogasawara was used as a photoresponsive 5 0 -cap demonstrating the impact of the distal aromatic ring on the dG Azo in the development of double-headed zebrash by controlling the expression of squint protein. 142

Spiropyran
Hirshberg and Fischer reported the rst photochemical reactions and photochromic phenomena of spiropyrans. 152 Spiropyrans are unique among the broad spectrum of photoswitches, due to the range of stimuli (e.g., temperature, visible light, mechanical forces, and solvent effects) able to induce its reversible isomerization. 153 Spiropyran consists of orthogonally orientated indoline and chromene moieties, joined by a quaternary carbon atom and thus is largely nonplanar.
Early work on spiropyran-modication on DNA oligonucleotides met with various obstacles including fast hydrolysis of spiropyrans in aqueous buffer solutions and the loss of the photoswitching ability in DNA. 6,154 This problem was largely alleviated when Heckel et al. incorporated spiropyran as a part of the DNA backbone using phosphoramidite chemistry and solid-phase synthesis (Fig. 18). 6 This photoswitch was also reported to work when incorporated at the 5 0 -end of homothymidine oligonucleotide in duplex DNA. 153 Reaction characteristics. Spiropyran groups incorporated as a linker in the phosphate backbone can undergo heterolytic cleavage of the C spiro -O bond either by thermal or photochemical perturbation (l max ¼ 365 nm). 6 Cleavage of the C spiro -O bond leads to the formation of the zwitterionic planar merocyanine due to the extended p-electron system (absorption around 400 nm). 6 This ring-opening accompanies a large change in dipole moment (Dm ¼ 7-15 D) and thus increases the overall polarity of the group. 155 The change is more pronounced than with other reversible photoswitches such as azobenzenes or diarylethenes. 6 The closed form can be regenerated by thermal energy or upon visible light irradiation (l max ¼ 530 nm). 6 The equilibrium in the photostationary state can be tuned both by the nature of the substituents or by the solvents used. 12 Notably, the spectral and photophysical properties of spiropyrans are tunable by changing the substitution pattern in a variety of positions. Different substitutions of spiropyran rings with different photophysical and thermodynamic properties have been reported. 6 Thermodynamic or structural characteristics. There is no melting temperature study for spiropyran included internally as a linker in the phosphate backbone. However, SP added at the end of an 8-bp (dT) 8 :(dA) 8 duplex showed that merocyanine (open-form) has a lower T m by 3-4 C compared with the spiropyran (closed form). 153 Another study showed that a nonreversible version of spiropyran modication (using click chemistry) in 17-bp DNA duplexes showed signicant destabilization (À12 to À20 C). 154 Biological applications. Not reported.

Diarylethene group
Diarylethenes (DAE) are known for excellent photochromic properties, such as negligible thermal relaxation, spectral tunability, and strong absorption bands upon photoconversion as well as high fatigue resistance against multiple photoswitching cycles. 156,157 Diarylethenes containing thiophene moieties and a cyclopentene ring are a special class of stilbenetype structures in which the ortho-hydrogens are substituted to suppress irreversible oxidation aer photocyclization of the cis isomer. Typically, the incorporated aryl rings are replaced by heterocycles to elongate the lifetime of the closed form, and the ethene moiety is oen embedded in a small ring to prohibit cistrans isomerization (Fig. 19). Diarylethene derivatives were rst introduced to oligonucleotides by Jäschke's group through 7-deazaadenosine. 158 Diarylethene under the irradiation of different wavelengths (250-370 nm) undergoes an electrocyclic rearrangement, generating strongly colored closed-ring isomers, whereas visible light (>400 nm) triggers the cyclo-reversion to the colorless opened-ring form which is thermally stable (Fig. 19). 158 Originally reported as a photoswitchable reaction in nonaqueous solvent, 158 the relatively low efficiency of photoisomerization in aqueous solvents had to be optimized using various substituents on the thiophene ring on 7-deazaadenosine which resembles purine 159,160 or on a (deoxy)uridine as a pyrimidine analog (Fig. 20). 161,162 In comprehensive testing of 13 different substituents on dU and dC nucleosides, dU with 2-pyridyl (2Py) and tert-butylester-phenyl (Ph t Bu) were found to be the best in the photoisomerization efficiency and thermal and photochemical stability (Fig. 20). 163 In particular, the photochromism (e.g., quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility) of the modied dU with 2-Py or Ph t Bu was maintained in the environment of the single-stranded oligonucleotide, and for Ph t Bu even in the duplex. These modications were also shown to be useful in controlling transcriptional activation. 163 Reaction characteristics. The characteristic absorption bands of the diarylethene chromophore at l ¼ 242 nm and l ¼ 305 nm are detectable in the UV/Vis spectrum of the modied nucleoside (colorless solutions-yellow). Irradiation by UV (250-370 nm) in the range of 5-30 min closed the diarylethene moiety and the visible absorption band rose at l ¼ 450 nm (strongly red). Closed isomers of DAE share the emergence of a broad absorption band between 400 and 600 nm with different maxima depending on the thiophene substitution extension of the conjugated system. 158 Other photophysical and chemical properties, such as isomerization wavelengths, quantum yields, thermal stability, and fatigue resistance can also be tuned by various substituents in the thiophene or cyclopentene ring. 162 The use of a broad range of light including low energy visible wavelengths is one of the strengths of DAE modication, which can be useful for biological applications.
Thermodynamic or structural characteristics. Incorporation of one uridine-caged diarylethene substituted with phenyl group in the thiophene ring decreased the T m of the DNA duplex by 2.3 C in both the open and closed ring forms compared with that of the unmodied duplex. 160 3 0or 5 0 -terminal modications were found to have a negligible effect on the stability in the open-ring form. 160 CD spectra of the same DNA, showed an apparent shi to a more A-like (i.e., RNA-like) conformation compared with natural DNA. The spectra were almost identical to unmodied DNA when the modication was terminal, and their UV-induced DNA conformational changes were also small. 160 Biological applications. In the study by Jäschke's group, a single diarylethene modication of dU R with 2-Py, Ph t Bu moieties positioned within T7 promoters were shown to modulate transcription rate in in vitro transcription assays. 163   The open-ring form containing promoters showed almost the same activity as the unmodied controls, whereas a $2 fold decrease in the transcription rate was observed for the closed form aer UV irradiation. 163

Concluding remarks
Photoconvertible groups offer a convenient way to alter molecular structures in a spatially and temporally controlled manner using light as the reaction initiator. Ideal photoreactive groups for biological applications would feature fast and complete photoconversions under mild, physiologically relevant conditions and would be capable of multiplexed, orthogonally controllable reactions (e.g., by choosing different wavelengths of trigger light). In recent years, signicant strides have been made in the availability and applicability of photoreactive oligonucleotides. Here, we compiled a list of currently available photoreactive groups for oligonucleotides to regulate DNA/RNA structure and function for diverse biological applications ( Table  1). The photoreactions are either irreversible (e.g., cleavage) or reversible (e.g., crosslink, isomerization, and intramolecular cyclization reactions), each with their own strengths but also limitations. For instance, reactions that use UV light may cause DNA or tissue damage and can interfere with the excitation/ emission of uorescent reporters used in in vitro/in vivo studies. Relatively moderate or low photoconversion yields (reaction completeness) of photoreactive groups also remain as hurdles. Expanding the array of available photoreactive modi-cations with enhanced photostability, biocompatibility and tunability would be exciting future directions. Continued research and development of light-convertible oligonucleotides promise to provide powerful tools for studying complex genetic mechanisms that uses DNA/RNA as their platforms including transcription, replication, and repair. For example, for DNA repair pathways such as NER where bulky lesions are recognized and repaired, o-nitrobenzyl, p-hydroxyphenacyl, and coumarin-related modications may be used as a bulky DNA lesion surrogate which can be readily switched on and off for various structural and mechanistic studies. Further improvements of photoconvertible nucleic acids to achieve higher photo-reaction efficiency and tunability of light at longer wavelengths may expand the applicability of photoreactions for biological investigations and modulation.

Conflicts of interest
There are no conicts to declare.