Modification of nucleic acids by azobenzene derivatives and their applications in biotechnology and nanotechnology.

FOCUS REVIEW DOI: 10.1002/asia.201402758

Modification of Nucleic Acids by Azobenzene Derivatives and Their Applications in Biotechnology and Nanotechnology Jing Li, Xingyu Wang, and Xingguo Liang*[a]

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Xingguo Liang et al.

Abstract: Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering. Keywords: azobenzene · DNA nanotechnology · nucleic acids · photoregulation · probes

Introduction Chemical modification of biomolecules has been widely carried out for modulating or tracking biological events, understanding biological mechanisms at the molecular level, and developing techniques such as DNA sequencing, biomolecule immobilization and so on. Molecules with catalytic activities, signal reporters, structural analogs, or other functional groups have been attached to biomolecules. In this field, chemical modifications of nucleic acids (NAs) have been most developed due to their automatic chemical synthesis on a DNA/RNA synthesizer.[1] Once the corresponding phosphoramidite monomer is synthesized, the modification can be easily achieved if a DNA/RNA synthesizer is available either in the laboratory or from a company supplying oligonucleotides. The significance and ongoing new findings of RNA also provide vast opportunities for modification of NAs.[2] Azobenzene, a typical azo compound, has a spatial size similar to a nucleobase pair,[3] and the azo group with its polarity lies between two highly hydrophobic benzene rings, just like the polarity distribution in a base pair. Modification of NAs by azobenzene is expected to stabilize the NA duplex (DNA/DNA, DNA/RNA, or RNA/RNA duplex), the basic NA structure. More interestingly, azobenzene can be used as a photoregulator due to its reversible photoisomerization, as the two structures of E (trans) and Z (cis) isomers differ significantly (Figure 1 a).[4] The trans form is

Figure 1. Basic structures of azobenzene (a) and azobenzene derivatives used for modifying nucleic acids (b). The photoisomerization is reversible, and the molecular size (C4–C4’ distance) changes between 0.90 nm (trans form) and 0.55 nm (cis form). R1 is the group for linking azobenzene derivatives to nucleic acids; R2 and R3 are usually groups to provide further functions. Two groups can be present at the same benzene ring. The distal benzene ring (with the R2 group) to the DNA/RNA backbone can also be replaced by a naphthalene ring. R1 and R2 are mainly used at the para position, although meta or ortho positions are also used in some cases.

planar and fits well between two base pairs in a DNA/RNA duplex, whereas the cis form is non-planar and causes great steric hindrance in the regular structure, preferring to flip out from the axis of the duplex. Thus, the introduced azobenzene can be used as a photoswitch for the formation of a NA duplex, and photoregulation of most functions of NAs can be expected.[5] On the other hand, azobenzene is the basic chromophore of widely used dyes and pigments, and it can be used as an ideal quencher for many fluorophores.[6] Accordingly, the applications of azobenzene-modified NAs may be very broad. This Focus Review highlights recent de-

[a] Dr. J. Li, Dr. X. Wang, Prof. X. Liang College of Food Science and Engineering Ocean University of China Nucleic Acids Chemistry and Biotechnology Laboratory No. 5 Yushan Road, Shinan-qu, Qingdao City (China) Fax: (+ 81) 532-82031086 E-mail: [email protected]

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(R2, R3 in Figure 1 b) have been introduced to adjust the maximum absorption wavelength of the trans form (e.g., SCH3, NACHTUNGRE(CH3)2),[6, 9] to enlarge the structural difference between trans and cis forms (e.g., ortho-CH3, para-isopropyl),[10, 11] or to improve the efficiency for quenching a fluorophore (e.g., NO2, NACHTUNGRE(CH3)2).[9, 12] When para-NACHTUNGRE(CH3)2 is present, the photoisomerization to the cis form becomes very difficult due to the fast thermal cis-to-trans isomerization (on the order of ms in water) and the overlap of the absorption peaks between trans and cis forms. The thermal cisto-trans isomerization can be suppressed to some extent at a higher pH or when an organic solvent is used in place of water.[13] Nishioka et al. found the very interesting phenomenon that the thermal stability of the cis form can be greatly increased for 4-carboxy-2’,6’-dimethylazobenzene as compared

velopments of azobenzene-modified NAs and their applications in biotechnology and nanotechnology.

1. Representative Structures of AzobenzeneModified Nucleic Acids 1.1. Structures of azobenzene derivatives used for modifying nucleic acids Basically, azobenzene and its derivatives are introduced into nucleic acids (NAs) for three purposes: 1) photoregulation of their biofunctions; 2) preparation of a probe for the detection of biomolecules; and 3) investigations on the mechanism of some enzymatic reactions related to NAs by using azobenzene as an internal probe. For different purposes, different derivatives should be selected, and the modification should be carried out using different approaches. In order to use facile synthesis procedures, azobenzene is usually attached through an amide bond. Thus, it is convenient if an amino or carboxyl group is present on the azobenzene (R1 in Figure 1 b). In some cases, azobenzene derivatives are simply attached at the end of a NA sequence with a linker. A small structural change on azobenzene can cause a large change in the absorption spectrum, photoisomerization efficiency, and the thermal stability of the cis form.[3a] Basically, an increase in conjugation causes a much bigger red shift of the absorption of the trans form than that of the cis form, accompanied by a great decrease in the thermal stability of the cis form. For example, when R1 in Figure 1 b is para-NH2, the maximum absorption wavelength of the trans form is around 355 nm, and the half-life of cis-azobenzene is about 1.0 h at 37 8C.[7] When COOH was used to replace NH2, the maximum absorption wavelength of the trans form shifts to about 325 nm, and the half-life of the cis form changes to 44 h.[8] For meta-NH2, the half-life of the cis form can be improved to 64 h.[7] Other substitute groups

X. G. Liang was born in Hebei Province, P.R. China in 1971. He received his M.Chem. from Tianjin Univ. in 1992. After3 years of teaching career in Tianjin Univ. he entered the Ph.D. course in Professor Makoto Komiyamas lab at The University of Tokyo, Japan. After only 2 and half years, he completed his Ph.D. in the summer of 2001. After 18 months Postdoctoral Research, he joined Prof. Maxim D. Kamenetskiis group at Boston University in 2003. In 2006, he was appointed as an Assistant Professor in the Department of Molecular Design and Engineering at Nagoya University, Japan, and then promoted to an Associate Professor with tenure in 2007. In 2011, he was appointed as a professor in Ocean University of China. J. Li was born in Shandong Province, P.R. China in 1983. She received her Ph.D. degree from Ocean University of China in 2012. After graduation, she was appointed as a lecturer in the College of Food Science and Engineering, and joined Prof. X. G. Liangs group. Her research interests lie in nanobiotechnology and the development of functional biomaterials based on DNA, RNA, etc.

Abstract in Chinese:

X. Y. Wang was born in Shaanxi Province, P.R. China in 1985. After 3 years of research in Prof. X. G. Liangs research group, he received the Ph.D. degree from Ocean University of China in June 2014. His research interests include applications of photoresponsive nucleic acids, biosynthesis of oligonucleotides, and developing DNA-detecting technologies using isothermal amplification approaches.

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with that of 4-carboxyazobenzene, whereas 4-carboxy-2,6-dimethlyazobenzene has a thermal stability similar to 4-carboxyazobenzene.[10, 14] They studied the mechanism of the effect of the alkyl group on the thermal stability and found that the steric hindrance is the main factor to retard the thermal cis-to-trans isomerization.[14] For an azobenzene derivative with a size longer than a base pair, the molecular design becomes more difficult if the stability of the NA duplex is required to be maintained.[11] This is especially the case for the photoregulation of DNA functions, where the difference between trans and cis form is important. Usually, when the azobenzene residue shows more flexibility in the NA duplex, the trans form cannot stabilize the duplex well, and the cis form will not destabilize it greatly.[15] As a result, the difference between trans and cis form becomes lower.

monomer can be synthesized so that the azobenzene can be introduced into any position of any sequence. Furthermore, multiple azobenzenes can be easily introduced. However, it is a challenge to retain the specific binding of a biomolecule at the modified site because the duplex structure changes greatly. For this case, both the backbone and side chain are quite different from the natural duplex. For Mode 3, no biofunction of DNA or RNA can be kept unless the azobenzene moiety is introduced at a position far away from the functional sequence.[16] 1.3. Structures of nucleic acids modified by attaching an azobenzene residue to natural DNA or RNA (Mode 1) Basically, modification of a biomolecule was carried out by simply attaching a functional group to the biomolecule, without changing its basic structure.[18] The recognition and binding of a biomolecule are considered to be very sensitive, and a small structural change may cause complete loss of its function. Thus, this approach to modify NAs has become popular as it does not require challenging efforts during the molecular design (Figure 3).[1] Keiper et al. introduced azobenzene at the 2’-position (Structure 1 in Figure 3) for modifying a DNAzyme to render it photoresponsive.[19] When the artificial DNAzyme 10–23 was modified in this manner, the catalytic activity was not affected in case the amide bond was attached at the ortho-position of azobenzene. By contrast, for the para-position, only 50 % activity remained. Asanuma et al. reported that para-azobenzene attached at the 2’-position through an ether bond (Structure 2 in Figure 3) showed the photoregulation ability for DNA duplex formation.[20] Attachment of azobenzene directly to the phosphorus atom through a phosphorothioate linkage (Structure 3 in Figure 3) was reported by Patnaik et al. in 2007.[21] The synthesis procedure is simple, and the azobenzene can be selectively introduced into any position with an iodo-alkyl agent once the phosphorothioate linkage is present. The phosphorothioate linkage can be simply introduced into the desired sites using tetraethylthiuram disulfide (TETD) in place of the conventional oxidant reagent I2. Basically, a DNA duplex is stabilized, especially when azobenzene is introduced between two dTs. The problem is that two diastereomers are present due to the chirality of the phosphorothioate linkage so that multiple modifications become troublesome. Azobenzene was also introduced through attachment to the amino group of adenine, and the base pairing was maintained so that in vitro selection involving azobenzene groups became possible (Structure 4 in Figure 3).[22] Mori et al. introduced the azobenzene residue into the 5-position of cytosine through an ethynyl group for photoregulation of DNA or RNA hybridization.[23] 4’-Carboxy-4-dimethylaminoazobenzene (Dabcyl) has been widely used as a universal quencher in a molecular beacon, which is a fluorogenic hairpin oligonucleotide probe.[6] It is generally attached at the 5’-end of an oligonucleotide through a C6 linker (Structure 6 in Figure 3). The

1.2. Modes of nucleic acid modification by azobenzene derivatives As shown in Figure 2, there are 3 modification modes to introduce azobenzene derivatives into a nucleic acid se-

Figure 2. Modes for introducing an azobenzene moiety into nucleic acids. Az signifies an azobenzene derivative; the arrows point out different positions to introduce an azobenzene residue. The linker in Mode 2 is usually a diol, which can be inserted into the DNA/RNA backbone by forming phosophodiester bonds. In Mode 3, the azobenzene is inserted into the backbone by forming phosophodiester bonds.

quence: 1) Modification can be carried out at the 2’-position of ribose, 5-position of thymine, or the phosphodiester linkage, etc.; 2) the (deoxy)ribose can be completely displaced by an artificial diol linker, on which the azobenzene is attached; and 3) the azobenzene can be directly introduced as the backbone of NAs, but not as part of the side chain.[16, 17] For Mode 1, structural perturbations by the modification are minimized, and the duplex structure does not change much compared to the natural one. One of the shortcomings of this approach is that difficult chemical procedures are usually required. For Mode 2, a universal phosphoramidite

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Figure 3. Structures of nucleic acids modified directly on the ribose, a base, or the phosphodiester linkage. The basic structure of the nucleotide is kept intact, and hydrogen bonds in base pairs are not affected greatly.

In this case, the DNA duplex can even be stabilized by the functional group such as azobenzene which acts as an intercalator.[27] This approach has the merit that the phosphoramidite monomer carrying the functional group can be synthesized so that the modification can be easily introduced at any position and even for multiple insertions either at one position or several positions. Figure 4 shows some structures of modified DNA in which an azobenzene derivative was tethered via an acylic scaffold. 2,2-Bis(hydroxymethyl) propionic acid was first reported as a 1,3-propanediol linker (abbreviated as C3-CO) for introducing azobenzene into DNA.[28] As 2,2-Bis(hydroxymethyl) propionic acid is a prochiral linker, two diastereomers

C6 linker can provide enough flexibility for Dabcyl to interact with the fluorophore. Certainly, the same structure can also be attached at the 3’-end.[24] Through a sulfate linkage, Dabsyl (dimethylaminoazobenzenesulfonic acid) can be attached at the 5’-end as Figure 4. Structures of Mode-2 linkers tethering azobenzene derivatives. a quencher with improved stability (Structure 8 in are obtained when the two hydroxyl groups are attached to Figure 3).[25] It can also be introduced within the sequence different groups. Fortunately, the two diastereomers of by attaching it to the 5-position of dT, leaving the 3’-termia short oligonucleotide (< 30 nt) can be easily separated by nus available for polymerase extension. HPLC.[28, 29] Azobenzene could also be introduced to PNA (peptide nucleic acids) for photoregulation of its hybridization with Natural DNA duplex itself is chiral: the typical B-form DNA and RNA.[26] Definitely, azobenzene can also be introDNA is a right-handed helix while Z-DNA is a left-handed helix. When the d-ribose is displaced by an acyclic diol, the duced into other nucleic acid analogs for various purposes. chirality of the linker is also important for stabilizing the duplex. When threoninol (C3-NH in Figure 4) is used as 1.4. Structures of nucleic acids modified by attaching an a linker, for example, the d-configuration fits well with the azobenzene residue to a non-ribose acyclic scaffold B-form DNA duplex and stabilizes the right-handed double (Mode 2) helix.[8, 17] Structural analysis by 2D NMR spectroscopy reSince the 1990s, a large number of acylic linkers have been vealed that the R-configuration of C3-CO (similar configuused for introducing functional groups.[17] The linker is usuration as d-threoninol) prefers to form the B-form righthanded duplex.[27a] For the S-configuration of C3-CO and lally a diol, which can be inserted into the NA backbone by forming phosphodiester bonds (Figure 2). An additional threoninol, the hydrophobic azobenzene can also intercalate substitute group is present for tethering the functional between two adjacent base pairs, and no obvious destabilizagroup. It is much more favorable when the functional group tion is observed. The higher structural stress due to the toris hydrophobic, planar, and with a size similar to a base pair. sion of the l- or S-configuration may cause a lower stability

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as compared with R- or d- form. It can be deduced that the l- or S-configuration should prefer the formation of ZDNA. When the modified DNA forms a duplex with a natural oligonucleotide, two cases can occur: 1) The base at the opposite position of azobenzene in the non-modified complementary strand forms a mismatch pair with the azobenzene moiety. In this case the two strands have backbones of the same length. Usually the duplex is destabilized in this case.[17, 30] 2) There is no nucleotide at the opposite position, and the non-modified complementary strand is shorter than the azobenzene-modified strand. Here, the azobenzene moiety is like a wedge to insert between two adjacent base pairs (Figure 5).[27a] Usually, trans-azobenzene can stabilize

Figure 6. Models of the asymmetric motif (A), interstrand-wedged motif (B), and azo-azo pairing motif (C). The long bars indicate trans-azobenzene moieties, while the short ones indicate nucleobases. Figure 5. Schematic illustration of a DNA duplex involving an azobenzene moiety. The trans-azobenzene residue intercalates between adjacent base pairs like a wedge, as determined by NMR spectroscopic analysis.[27a]

with that of the native DNA duplex (Figure 6 a).[31] In this case, the structure was highly asymmetric, and the length of the modified strand is 29 nt, which is 9 nucleotides longer than its natural complementary strand. However, when more azobenzenes were introduced, the duplex became unstable.[31, 32] In case that it is not necessary to target natural DNA or RNA, azobenzene residues can be introduced into both strands. As shown in Figure 6 b, an artificial duplex can be built with the ratio of azobenzenes to base pairs being 1:1.[33] The symmetry is greatly improved, although the azobenzene also inserts like a wedge and the structure is locally asymmetric. Interestingly, this artificial duplex is much more stable than the native duplex of the same sequence. One azobenzene stabilizes the duplex even more than an A-T pair, but less than a G-C pair.[33b] NMR analysis proved that hydrogen bonds in a base pair formed tightly between two azobenzene molecules.[33b] The very strong negative–positive cotton effect indicates that a regular right-handed duplex was formed. More interestingly, the recognition ability between two complementary strands is even higher than that of the native duplex, and the mismatch between two azobenzenes causes a greater decrease in the melting temperature (Tm).[33b] Furthermore, duplex formation is also possible at an azobenzene-to-base pair ratio of 2:1 (Figure 6 c).[32] In this artificial duplex, two azobenzenes form an azo–azo pair composed of two azobenzenes from different strands. Obviously, the structure is completely symmetric at the region in which azobenzenes are present. The structure obtained from molecular modeling is a highly symmetric duplex.[32] Interest-

the duplex because the destabilization effect caused by the steric torsion is compensated by the strong stacking of azobenzene with base pairs.[28] When a short C2 linker is used (C2-CO and C2-O in Figure 4), a more stable duplex can be obtained, although the improvement is not so notable.[15] These results demonstrate that the DNA duplex is structurally quite flexible, and even a drastic structural change is allowable for its modification (Figure 5). Modification at the para position on the distal benzene causes an increase in length of azobenzene. As mentioned previously in this review, azobenzene has the exact size to fit with base pairs. Therefore, the presence of a bulky substitute group at the para position causes steric hindrance with the backbone of the complementary strand so that the stability is greatly decreased.[11] As shown in Figure 5, almost no space is left for bulky groups, and even a methyl group can cause destabilization. 1.5. Modes of duplex formation using Model 2-modified oligonucleotides bearing multiple azobenzenes Introduction of multiple azobenzenes by a chiral linker (not a pro-chiral linker like C3-CO) is also possible. With dthreoninol as a linker, 9 azobenzene moieties were introduced into a 20 bp duplex (the ratio of base pairs to azobenzene is 20:9), and the stability did not decrease as compared

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a DTm of 36.2 8C. However, the strong interaction between base pairs and azobenzene decreased greatly the efficiency of cis-to-trans photoisomerization. For example, only about 25 % of azobenzene could be isomerized to the cis form at 10 8C.[35b] The large difference in structure between the trans and cis form can explain the photoregulation mechanism: the intercalated hydrophobic planar trans-azobenzene stabilizes the duplex or triplex by stacking interaction, whereas the nonplanar cis-azobenzene destabilizes the duplex or triplex by steric hindrance.[27] Improvement and diversity of the photoregulation by chemical modification. For applications of photoregulation of biofunctions by using azobenzene-modified NA, the following problems need to be solved: 1) The thermal stability of the cis form is insufficient. For example, in the case of azobenzene tethered to DNA by C3-NH, the half-life of cisazobenzene at 37 8C is only about 1 h; 2) The photoregulation efficiency is too low. For example, only 90 % of the cis form can be obtained in the single-stranded state. In the case of double-stranded state, the fraction of the cis form is even less than 30 %, especially at a lower temperature. Another reason for the low regulation efficiency is that the DTm caused by trans-cis photoisomerization is not large enough; 3) The wavelength for trans-to-cis isomerization is not long enough (< 360 nm), and the cells are easily damaged at irradiation below 360 nm; 4) A significant change in the duplex structure hampers the interaction of DNA with other biomolecules; and 5) The azobenzene moiety cannot be introduced using DNA and RNA polymerase because the corresponding triphosphate of the azobenzene monomer cannot be used as the substrate. Chemical modification and an improved molecular design may satisfy the above requirements (Figure 8). As mentioned in Section 1.1, the thermal stability of the cis form can be improved by using C3-NH to replace C3CO and by introducing two methyl groups at the ortho-positions of the N=N bond on the distal benzene ring. The photoregulation efficiency was also improved by using 2’,6’-dimethyl azobenzene (2’6’-Azo).[10, 14] For a 12 bp-long DNA duplex, the DTm was improved from 5.7 8C to 14.6 8C by replacement of azobenzene (p-Azo) with 2’6’-Azo (see Figure 8 for its structure). However, the trans-to-cis isomerization becomes more difficult, and only 65–70 % of 2’6’-Azo can be isomerized to the cis form at most.[10] The photoregulation efficiency could be further improved by introducing multiple azobenzenes. The DTm was improved to more than 60 8C by using 9 azobenzenes in a 20 bp-long duplex.[31] However, no obvious improvement could be obtained when 9 moieties of 2’6’-Azo were used to replace 9 moieties of pAzo due to the lower trans-to-cis photoisomerization efficiency.[36] The photoregulation efficiency was further improved by using the artificial duplex as shown in Figure 6 b and 6 c. Almost no duplex can form in the cis form, and complete ON–OFF photoregulation became possible.[32, 33] When a large substituting group is present in the para position of the distal benzene ring, interestingly, cis-azoben-

ingly, the stability of this duplex is also very high, and an azo–azo pair can stabilize the duplex like a G-C pair. This result showed that the recognition ability between nucleobases could be utilized to construct artificial materials with a regular structure. It sheds new light on nanotechnology, demonstrating that parts of biomolecules can be combined with functional groups generated through organic synthesis. The detailed structures have been investigated by NMR spectroscopy and molecular modeling. Fujii et al. showed that two azobenzenes stack with each other in an antiparallel configuration.[9] The azo–azo pair can also form in an RNA/RNA duplex, in which the positions of these two azobenzenes are reversed.[34]

2. Applications of Azobenzene-Modified Nucleic Acids 2.1. Photoregulation of DNA or RNA secondary structures Photoregulation of DNA hybridization by using azobenzene-modified DNA. The basic secondary structure of nucleic acids (NAs) is the double helix. If the duplex formation and dissociation can be photoregulated, photoregulation of most functions of NAs becomes possible.[27] The first example reported is the evaluation of the photoregulation ability of an azobenzene tethered to a C3-CO linker (see Figure 4) in an 8 bp-long DNA duplex.[27b] The Tm measurement results showed that DTm (the difference in Tm between trans and cis forms) was 8.9 8C. Through estimation of the potential photoregulation efficiency it was assumed that 10–20 % of the duplex could newly open (e.g., 60 % duplex formed under visible light, and 45 % formed under UV light) by irradiation with UV light at a fixed temperature.[27b] Amazingly, this study showed that photons could be used to trigger the dissociation of a DNA duplex reversibly (Figure 7. Basi-

Figure 7. Schematic illustration of the photoregulation with light of various wavelengths by using an azobenzene-modified NA.

cally, for all short sequences investigated (16 types of duplexes with NXN, N = A,G,C,T), the photoregulation proved possible, although the DTm changed with sequence to some extent.[29, 15] When the modified DNA was used as the third strand in a DNA triplex, DNA triplex formation and dissociation were also reversibly photoregulated.[35] One azobenzene tethered to a C3-CO linker in the middle position of a 13nt-long triplex-forming oligonucleotide (TFO) could cause

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provement basically comes from the nature of this kind of chemical bond but not from a weaker stacking with base pairs. A DNA/RNA duplex can be photoregulated efficiently when azobenzene is tethered to the DNA strand in a manner similar to that of the DNA/DNA duplex.[40] Azobenzene can also be introduced into RNA for photoregulation of a DNA/ RNA or RNA/RNA duplex. In some cases, even a larger DTm can be obtained as compared with that of the DNA/DNA duplex.[41] However, the Tm Figure 8. Various azobenzene derivatives investigated to improve the photoregulation efficiency. values of the azobenzene-modified DNA/RNA, RNA/RNA, zene forms a much more stable duplex than the trans and RNA/DNA duplexes are similar or lower than the form.[11] For a 12 bp duplex, the Tm value of the cis form was values of the corresponding native duplex, probably because it is difficult for intercalators to stabilize the A-form NA higher than the trans one by 6.7 8C when Ip-Azo was used duplex. The larger DTm is mainly caused by the destabiliza(Figure 8). This reverse photoswitch effect can be explained as follows: the alkyl group in the cis form binds to the surtion effect of the cis form. face of the major groove of the B-type DNA duplex by hyAzobenzene attached to bases or 2’-OH also shows a phodrophobic interactions; in the trans form, however, the big toregulation ability, albeit weaker as compared to that of substituting group causes large steric hindrance towards the azobenzene attached to the non-ribose acyclic linker.[20] deoxyribose of the other strand (see Figure 5 for the NMR Mori et al. synthesized modified DNA with azobenzene atstructure of the trans form). tached to C5 of deoxyuridine via an ethynyl group and For azobenzene derivatives tethered to C3-CO or C2-CO, showed that it could preferably photoregulate DNA/RNA cis-azobenzene could only be obtained with a high yield by duplex formation as compared with DNA/DNA duplex forirradiation with UV light at a wavelength shorter than mation.[23] 380 nm. In order to reduce the possible harm to biological Wu et al. reported that a hairpin DNA structure can be molecules and to improve the diversity of photoregulation, photoregulated by using 4,4’-bis(hydroxymethyl)-azobenlight with a wavelength longer than 400 nm is favorable. zene inserted directly into the DNA backbone (Figure 2, When dimethylthiomethylazobenzene (DMS-Azo, Figure 8) Mode 3).[42] When the photoregulation strategy is used to was used, light of 400 nm could photoisomerize the derivamodulate the formation of a hairpin structure, it can also be tive to the cis form, and the DTm of the 12 bp duplex was as called a photoresponsive molecular motor, although this term can be considered exaggerated in this context.[43] large as 12 8C.[34, 37] In addition, t1/2 of cis-DMS-Azo attached to an oligonucleotide is 6.4 h at 60 8C, which is longer than McCullagh et al. investigated an azobenzene-capped DNA that of non-substituted azobenzene.[37] For m-Nap, which hairpin coupled to an atomic force microscope (AFM) tip, showing that the photoinduced trans-to-cis isomerization of can be isomerized to the cis form with light of 390 nm, the azobenzene affects both the overall length of the molecule efficiency of photoregulation was not improved due to the and the ability of the DNA bases to hybridize; they named low photoisomerization efficiency and the smaller DTm betheir system as DNA-based optomechanical molecular tween trans and cis forms. However, the photoregulation efmotor.[44] ficiency of DNA triplex formation can be greatly im[38] proved. The trans-to-cis photoisomerization in a duplex state 2.2. Photoregulation of bioreactions using azobenzenecould be improved by using R-glycerol (C2-O, Figure 4) as modified nucleic acids a linker, and an ether (R-O-Ar) bond was used to attach azobenzene. For example, at 25 8C, a high percentage of azoPhotocontrol of enzymatic reactions by using azobenzenebenzene (75 %) in a 20 bp-long duplex was isomerized to modified DNA. Azobenzene-modified DNA carrying the the cis form.[39] Even at 0 8C, up to 67 % of azobenzenes C3-CO linker was first used to photoregulate DNA primer extension by DNA polymerase. (Figure 9 a).[45] Azobenzene could still be isomerized to the cis form, which is much higher than that obtained for a d-threoninol-tethered one was introduced at the 5’-end of a short oligonucleotide, (only about 30 % at 25 8C). It can be deduced that the imwhich could hybridize to a DNA template. For the trans

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azobenzene was introduced into the arm of 8-17 DNAzyme because more RNA substrate could bind to the DNAzyme. When two p-Azo moieties (Figure 8) were present at the catalytic loop, however, the cis form showed a higher cleavage activity, thus indicating that the structural change caused by trans-azobenzene decreased the cleavage activity more.[47] Similarly, Keiper et al. introduced para-, meta-, and ortho-azobenzene into the 2’-position of a thymidine (structure 1 in Figure 3) in 10-23 DNAzyme and found that the cis form showed a several-fold higher activity than the trans form.[19] No obvious difference in photoregulation efficiency was found for the three isomers. When azobenzene was introduced into the catalytic loop, the structural change between trans and cis forms but not the formation and dissociation of a duplex caused the difference in activity. Liang et al. also showed that when azobenzene-modified DNA was used as the template for DNA ligation by T4 DNA ligase, cis-azobenzene had a higher activity.[48] Interestingly, a high yield of ligation was obtained in the presence of azobenzene at the ligation site. Photoregulation of transcription. Transcription by T7 RNA polymerase was photoregulated by using an azobenzene-modified T7 promoter (Figure 10).[13a, 49] Here, no comFigure 9. Photoswitched DNA primer extension (a) and RNA cleavage by RNase H (b). The design of trans-OFF and cis-ON is important here because the efficiency for turning off the reaction is the key point to give the clear-cut photoregulation.

form (< 2 % in cis form), the modified DNA formed a stable duplex with the template, and the primer extension stopped at the position of the 5’-azobenzene (OFF); for the cis form (~ 50 %), the primer extension could go through to the end of the template (ON). Because trans-to-cis photoisomerization greatly decreases (usually < 50 %) when transazobenzene intercalates between base pairs in a duplex, the molecular design to achieve trans-OFF and cis-ON is important for clear-cut photoregulation. For the similar reason, trans-OFF and cis-ON was also designed for the photoregulation of RNA digestion by RNase H (Figure 9 b).[46] The native antisense DNA dissociates from the photoresponsive DNA duplex after irradiation with UV light (cis form) and hybridizes to the target RNA so that RNase H can cut it. For the trans form, the more stable duplex forms and the digestion is greatly decreased. Partial dissociation of the native antisense DNA from azobenzene-modified DNA caused by UV-light irradiation is sufficient for its hybridization with the sense RNA and its cleavage by RNase H. However, even in this interesting design, a complete OFF state is difficult to obtain because a small amount of DNA can bind to RNA and cause cleavage in a rapid turnover manner. Photoregulation of RNA cleavage by a DNAyzme was also realized by introducing azobenzene into the sequence of the DNAzyme. Liu et al. reported that the trans form showed a higher cleavage activity than the cis form when

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Figure 10. Azobenzene-modified T7 promoter for photoregulation of gene transcription.

plete and permanent dissociation occurs during transcription, and the structural change upon photoisomerization is the key point for efficient photoregulation. Interestingly, when only one azobenzene was attached, only a two-fold increase was observed for trans-to-cis photoisomerization. When two azobenzene moieties were attached at positions 9 and 3 (Figure 10), the difference increased to 6–10-fold due to a synergistic effect.[49a] The photoregulation mechanism can be explained as follows: In the trans form, the azobenzenes intercalate between base pairs, thus resulting in large structural changes, and T7 RNA polymerase cannot bind to start the transcription. In the cis form, the azobenzenes prefer to flip out from the duplex so that the structure is close to the non-modified one, and the transcription activity recovers. Transcription by SP6-RNA polymerase could be photoregulated similarly.[50] The detailed mechanism of photoregulation was also clarified by introducing two kinds of azobenzenes (p-Azo and 2’,6’-azo, see Figure 8) with different thermal isomerization rates of the cis form to prepare the cis–trans (i.e., the azo-

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benzene group at the 9 position is in the cis form and the other one at the 3 position is in the trans form), trans–cis, and cis–cis species.[51] The results showed that cis–cis is the most active species for transcription. The trans-to-cis isomerization at the 9 position mainly causes an increase in the affinity (smaller Km) of T7 RNA polymerase, and the transto-cis isomerization at the 3 position mainly causes an increase in the reaction rate (larger kcat).[51] It is surprising that, although two azobenzene moieties are present, the rate of transcription with the cis–cis form promoter is not decreased much as compared with that with the non-modified, native promoter. A photoresponsive GFP gene was also constructed by attaching the azobenzene-modified T7 promoter.[49e] Translation of GFP protein with an in vitro gene expression system could be switched ON by irradiation with UV light (~ 340 nm).[49e] 2.3. Photoresponsive nanostructures or nanodevices Recently, DNA nanostructures have been constructed such as DNA origami, nanosoccer balls, or even well-designed nanomachines with open-close, rotation, and transportation functions. Usually, no enzymatic reaction is required, and azobenzenes can be introduced into any positions for constructing photoresponsive DNA nanodevices. Furthermore, photoswitching of bioreactions can be achieved by attaching the nanostructures to some functional NAs. Photoresponsive nanostructures. Some 2D and 3D nanostructures are constructed based on the hybridization of 4– 6 nt sticky ends of basic parts. Accordingly, photoresponsive structures can be constructed simply by adding several azobenzenes to the sticky ends. In the trans form, the nanostructure forms like the native DNA; it can break into pieces after photoisomerization to the cis form. As shown in Figure 11 a, a light-sensitive nanometer-sized soccer ball was made based on this strategy.[52] For each 4 nt sticky end, two azobenzenes were introduced, and a symmetric 4 bp duplex with 4 azobenzenes forms after assembly (see Figure 6 c for the structure of the trans form). The formed nanostructure was observed by AFM imaging (Figure 11 b). After irradiation with UV light for 30 s, the nanoball was broken, as recorded by the high speed of a real-time AFM.[52] A high photoregulation efficiency was realized because hundreds of azobenzenes were present in one nanostructure. A cascade effect can be expected: after initiation of the destroying process, more and more azobenzenes can be photoisomerized to the cis form so that the nanostructure collapses completely. One-dimensional photoresponsive DNA-templated protein arrays using azobenzene-modified DNA duplex as scaffold were also constructed.[53] The fluorescent protein arrays could be modulated by UV-light irradiation due to the dissociation of the scaffold duplex. The authors of this study claimed that complex nanobiotechnological devices may be constructed by using this concept. Photon-driven DNA nanomachines. Based on strand displacement or strand migration, numerous DNA nanodevices

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Figure 11. A photoresponsive DNA nanosoccer ball involving azobenzene-modified sticky ends. a) The molecular design of the sticky ends. b) AFM image of nanoballs under visible light. The scale bar in the inset is 100 nm. The diameters of these nanoballs range from 50 to 150 nm.

have been made such as DNA walkers, DNA tweezers, and DNA motors. DNA oligonucleotides are usually used as the fuel to drive these devices by hybridization and/or dissociation of a DNA duplex. When azobenzene-modified oligonucleotides are used instead of native DNA, the nanodevices can be simply operated by light, a clean energy source. Figure 12 a shows one model of photoresponsive nanomachines.[54] The operations of this photoresponsive DNA tweezer were easily repeated by alternating irradiation with UV light and visible light. A single-molecule DNA nanomachine was also constructed to cleave RNA.[55] The RNA cleavage activity of a responsive DNAzyme can be reversibly controlled by light (Figure 12 b). Photoresponsive ribozymes were also constructed by using the same strategy.[55b] Another merit of using azobenzene-modified oligonucleotides to construct a nanomachine is that more complex operations are possible by the combination of various azobenzene derivatives (Figure 8). Figure 12 c shows a light-driven DNA seesaw involving both p-Azo and DMS-Azo.[56] Under irradiation with light of 450 nm, both p-Azo and DMS-Azo are mainly present in the trans form; under irradiation with light of 370 nm, both of them mainly exist in the cis from. Interestingly, p-Azo is present in the cis form under 340 nm light but in the trans form under 390 nm light; and DMSAzo is in cis form under 390 nm light but in the trans form under 340 nm light. Thus, the four states as shown in Figure 12 became possible.

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Figure 12. Photoresponsive DNA nanomachines. a) Photon-driven DNA tweezers; b) Photoswitched DNAzymes by photoregulating the topological structure of a DNAzyme; c) A DNA seesaw driven by light of various wavelengths by using both p-Azo and DMS-Azo (Figure 8), which are isomerized to the cis form by light of 330–380 nm and 380–420 nm, respectively.

Other light-driven DNA nanostructures. Azobenzenemodified DNA has also been used in DNA origami technology. The hybridization and dissociation of azobenzene-modified DNA was directly observed by Endo et al. using AFM.[57] The authors constructed a DNA origami planar structure with a hole in the middle in which two parallel DNA duplexes were present. An azobenzene-modified DNA duplex was designed to bridge the above two parallel duplexes (Figure 13 a). When the modified duplex was formed, the parallel duplexes were drawn close to form an X-shaped structure. Dissociation after irradiation with UV light resulted in the recovery of the parallel structure. Other DNA origami structures including 3D structures can become photoresponsive by using azobenzene-modified NAs. For example, one side of a cubic structure can be opened and closed by light irradiation using azobenzenemodified DNA duplex as the photoswitch. Tans group reported several applications of photoresponsive DNA. They combined silver nanostructures and photo-

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Figure 13. Light-driven nanostructures and nanomotors using azobenzene-modified DNA. a) Bridging and release of two dsDNAs by formation and dissociation of an azobenzene-modified duplex DNA; b) Lightmodulated DNA tetrahedral structures; c) Light-driven caps for drug release from pores of silica nanoparticles; d) Photoswitching of an assembly of gold nanoparticles.

regulation of DNA hybridization to construct an efficient photosensitive DNA nanomotor.[58] Silver nanoparticles in

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an oscillating electromagnetic field will lead to field amplification both inside and in the near-field zone outside the particles. The authors claimed that the spectral overlap between azobenzene absorption and plasmonic resonances of silver nanowires increased the close-open conversion efficiency to 85 %. By using azobenzene-modified DNA, they also constructed photoresponsive DNA tetrahedra which can be controlled by light (Figure 13 b).[59] Basically, for many DNA nanostructures, photoresponsiveness could be realized simply by replacing some DNA strands with azobenzenemodified ones. Azobenzene-modified DNA was also used to construct a photoresponsive cap that was immobilized at the pore mouth of mesoporous silica nanoparticles, and the drug inside the nanoparticles could be released by UV-light irradiation (Figure 13 c).[60] According to the authors, this system could find applications in cancer therapy. This research widens the application of azobenzene-modified NAs to other nanostructures either constructed from biomolecules or other materials. For example, light-responsive DNA-functionalized gold nanoparticles were prepared by attaching azobenzene-modified oligonucleotides (Figure 13 e).[61] These particles exhibited reversible photoswitching of their assembly behavior. Exposure to UV light results in dissociation of the DNA duplex and nanoparticle assembly is destroyed. The isomerization is reversible upon exposure to blue light, resulting in rehybridization and reassembly of the DNA-linked nanoparticle clusters. The assembly and dispersion can be observed by the naked eye (Figure 13 d). Interestingly, the authors even showed that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and thus the photon dose can be used in place of temperature or ionic strength to control the hybridization stringency, with the ability to discriminate single-base mismatches (SNPs discrimination). Based on the above molecular designs, it can be expected that azobenzene-modified NAs hold great potential for applications in nanotechnology. Several photoresponsive aptamers were obtained by in vitro selection or direct molecular design.[22, 62] Azobenzenemodified DNA was used to control thrombin activity by changing the structure of the thrombin aptamer.[62a] In the trans form, the azobenzene-modified part hybridizes with part of the aptamer so that it cannot form the correct structure to bind to thrombin. After isomerizing azobenzene to the cis form, the structure of the aptamer recovers and the binding by the aptamer inhibits the interaction between thrombin and its substrate. Accordingly, the activity of thrombin can be simply regulated at a selected point and time through light illumination (Figure 14). Liu et al. selected an RNA aptamer in vitro from a random sequence library of RNAs with azobenzene residues. The aptamer bound to hemin more strongly in the trans form than in the cis form.[22] Besides establishing a light switch, this research also showed that functional molecules could be introduced during in vitro selection for finding aptamers with new functions.

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Figure 14. Illustration of a strategy for the photoregulation of thrombin activity using a reversible photoswitchable azobenzene-modified aptamer probe.

2.4. Other applications of azobenzene-modified DNA. As the quencher in a molecular beacon. As mentioned previously in this review, azobenzene derivatives can be used as a universal quencher in a molecular beacon.[6] A molecular beacon is a fluorogenic hairpin oligonucleotide probe with a fluorophore/quencher pair that can be used for quantitative polymerase chain reaction (PCR), pathogen detection, and other detections of a specific nucleic acid sequence. In the closed state, the fluorophore is very close to the quencher, and almost no fluorescence can be observed. When the target molecules are present, the fluorophore separates from the quencher and gives strong fluorescence emission. The first quencher used in a molecular beacon was (4-(4-dimethylaminopheynlazo) benzoic acid (Dabcyl), which was attached to the end of an oligonucleotide (Figure 3). Asanuma et al. developed an “in-stem molecular beacon”, in which both fluorophore and quencher are introduced and dimerized at the middle of the stem but not at the end (Figure 15).[12] The acylic linker is favorable for introducing both fluorophore and quencher within the sequence (see Figure 6 c for the azo-azo pairing style). This design can significantly lower the background fluorescence due to the strong interaction between the fluorophore and the quencher within the base pairs. Using this design, the introduction

Figure 15. In-stem molecular beacon with a diol linker. a) Basic principle of an in-stem molecular beacon; b) Multiple fluorophore/quencher pairs in a molecular beacon. The longer bars and ovals indicate trans-azobenzene residues (quencher) or fluorophores, and the short ones indicate nucleobases.

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of multiple fluorophore–quencher pairs also becomes possible (Figure 15 b).[63] Interestingly, even for the azobenzene derivatives with a maximum absorbance shorter than 400 nm, such as p-Azo or S-Azo (see Figure 8 for molecular structures), a strong quenching effect was observed. These azobenzene-bearing probes could possibly be used in vivo, and the genotoxicity could be avoided by proper molecular design.[64] As an internal probe. The effect of a local structural change of biomolecules due to the introduction of azobenzene (only p-Azo attached on d-threoninol was discussed in this section, Figure 8) can also be used as an “internal probe” to ascertain detailed information on the interaction between NAs and other biomolecules. For example, in the photoresponsive T7 promoter (Figure 10), the change of transcription activity with the position of azobenzene provides information on the interaction between the promoter and T7 RNA polymerase.[49a, 51] The fact that introduction of cis-p-Azo at the 3 and 9 position does not affect the transcription much indicates binding of T7 RNA polymerase to the major groove, but not the minor groove, because structural analysis showed that cis-azobenzene flips out to the minor groove.[51] The difference between the trans and cis forms can also yield valuable structural information. Only when two trans-azobenzenes were introduced did the transcription activity decrease greatly. This fact indicates that the structural change caused by the interaction of one azobenzene is not big enough to prevent the binding of T7 RNA polymerase to the T7 promoter.[49] When an azobenzene-modified DNA was used as the template for DNA ligation and azobenzene was introduced at exactly the opposite position of a nick site, T4 DNA ligase could carry out DNA ligation with a high yield.[48] This indicates that a large structural change at the activity site of T4 DNA ligase was allowed for DNA ligation. More interestingly, when azobenzene-modified DNA was used as the template for primer extension, Tth DNA polymerase could overcome the barrier of the azobenzene molecule to give the full-length DNA product.[48] For other DNA polymerases, the primer extension stopped at the position of azobenzene.[48] Obviously, the structure at the activity site of Tth DNA polymerase is quite different from that of other DNA polymerases. By introducing azobenzene into a modified DNA, photoregulation of RNA cleavage by LuIII ion aided by an intercalator (e.g., acridine residue) was also realized.[65] The transazobenzene impeded the interaction of acridine so that RNA cleavage was slowed down, and isomerization to the cis form decreased the retarding effect because cis-azobenzene could flip out more easily. Azobenzene was also introduced into siRNA to suppress the off-target effect of RNAi.[66] The possibility of RNA-induced silencing (RISC) complex formation with the non-target strand was greatly decreased due to the steric hindrance. This result indicated further that the introduced azobenzene did not affect the RNAi process, despite the large structural change. Based on this knowledge, Kamiya et al. introduced a fluorophore/

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quencher pair (with Dabcyl as the quencher and thiazole orange (Cy3) as the fluorophore) to label siRNA.[67] Other applications include the preparation of a photoresponsive DNA gel using azobenzene-modified DNA[68] and mechanistic studies on coherent clustering of azobenzene derivatives with various substituting groups within a DNA duplex.[9] Two or several molecules could be designed to interact with each other within a NA duplex.[37] These studies can extend the applications of azobenzene-modified NAs to basic studies on the mechanism of the interaction between molecules at a precisely controlled level.

Conclusions In conclusion, azobenzene derivatives can be used as a photoswitch, quencher, or internal probe to modify nucleic acids. Azobenzene-modified nucleic acids can be applied for photoregulation of their functions, construction of DNAbased nanostructures and nanodevices, probes for nucleic acid detection, as well as for understanding the interactions between biomolecules. Although further improvements of azobenzene-modified nucleic acids are required, such as lower toxicity, higher photoregulation efficiency, and longer wavelength of light for irradiation, their applications in vivo are promising because they are much more difficult to be digested by nuclease than natural nucleic acid. For applications in nanotechnology, the combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering.[69]

Acknowledgements This work was supported by “Fund for Distinguished Young Scholars” of Shandong province [JQ201204], Program for Changjiang Scholars and Innovative Research Team in University [IRT1188], “Wan Ren Plan of Shandong Province” and “National Youth Qianren Plan”.

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FOCUS REVIEW Only light can do that: By using transcis photoisomerization, DNA duplex formation and dissociation could be photoregulated simply by light irradiation. Based on this basic idea, further improvements of azobenzene-modified nucleic acids and their applications were developed. By combining functional groups with biomolecules, novel nanomaterials can be developed and may contribute greatly to DNA nanotechnology.

DNA Nanotechnology Jing Li, Xingyu Wang, Xingguo Liang*

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Modification of Nucleic Acids by Azobenzene Derivatives and Their Applications in Biotechnology and Nanotechnology

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Nucleic acids nanotechnology.

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Nanotechnology for delivery of peptide nucleic acids (PNAs).

Palladium(II)-coproporphyrin I as a photoactivable group in sequence-specific modification of nucleic acids by oligonucleotide derivatives.

DNA materials: bridging nanotechnology and biotechnology.

Potential biological applications of bio-based anacardic acids and their derivatives.

Frontiers in Nucleic Acid Nanotechnology.

Chemistry enters nucleic acids biology: enzymatic mechanisms of RNA modification.

Synthesis of peptide nucleic acids containing pyridazine derivatives as cytosine and thymine analogs, and their duplexes with complementary oligodeoxynucleotides.

Structure-property relationships of symmetrical and asymmetrical azobenzene derivatives as gelators and their self-assemblies.

Guest editorial: nucleic acid nanotechnology.

Presence of human immunodeficiency virus nucleic acids in wastewater and their detection by polymerase chain reaction.

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Nanotechnology applications in medicine and dentistry.

A survey of advancements in nucleic acid-based logic gates and computing for applications in biotechnology and biomedicine.

Atherosclerosis and Nanotechnology: Diagnostic and Therapeutic Applications.

Applications of biotechnology in nutrition.

Circulating nucleic acids: An analysis of their occurrence in malignancies.

Nanotechnology applications in thoracic surgery.

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