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• W3107524262 abstract "•The first high-affinity monoclonal antibody specific for a popular photocaging group•A new tool for selective detection of DMNB-tagged proteins in complex mixtures•Enables non-covalent capture of native proteins with surface-exposed DMNB groups•Orthogonal protein manipulation by photocage-selective capture and photolytic release Photochemical transformations enable exquisite spatiotemporal control over biochemical processes; however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. Here we created a high-affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues, which were used for raising a high-affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a variety of DMNB-caged target proteins in complex biological mixtures. This combined orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays. Photochemical transformations enable exquisite spatiotemporal control over biochemical processes; however, methods for reliable manipulations of biomolecules tagged with biocompatible photo-sensitive reporters are lacking. Here we created a high-affinity binder specific to a photolytically removable caging group. We utilized chemical modification or genetically encoded incorporation of noncanonical amino acids to produce proteins with photocaged cysteine or selenocysteine residues, which were used for raising a high-affinity monoclonal antibody against a small photoremovable tag, 4,5-dimethoxy-2-nitrobenzyl (DMNB) group. Employing the produced photocage-selective binder, we demonstrate selective detection and immunoprecipitation of a variety of DMNB-caged target proteins in complex biological mixtures. This combined orthogonal strategy permits photocage-selective capture and light-controlled traceless release of target proteins for a myriad of applications in nanoscale assays. Optical control of protein function enables an exquisite spatial and temporal interrogation of biological processes that cannot be accomplished with other conditional control elements. This is often achieved by using photolabile protecting groups (caging groups); removal of these caging groups by light (decaging) is a mild and noninvasive technique that is completely orthogonal to other chemical processes in a biological system. This approach has been successfully applied for photochemical control of the activity of small molecules, peptides, oligonucleotides, and proteins (Ankenbruck et al., 2018Ankenbruck N. Courtney T. Naro Y. Deiters A. Optochemical control of biological processes in cells and animals.Angew. Chem. Int. Ed. 2018; 57: 2768-2798Crossref PubMed Scopus (204) Google Scholar; Dumas et al., 2015Dumas A. Lercher L. Spicer C.D. Davis B.G. Designing logical codon reassignment – Expanding the chemistry in biology.Chem. Sci. 2015; 6: 50-69Crossref PubMed Google Scholar; Shimizu et al., 2006Shimizu M. Yumoto N. Tatsu Y. Preparation of caged compounds using an antibody against the photocleavable protecting group.Anal. Biochem. 2006; 348: 318-320Crossref PubMed Scopus (4) Google Scholar; Spicer and Davis, 2014Spicer C.D. Davis B.G. Selective chemical protein modification.Nat. Commun. 2014; 5: 4740Crossref PubMed Scopus (581) Google Scholar). Installation of photocaging groups in biomolecules can be achieved by (1) chemical or chemo-enzymatic modification of native biopolymers or (2) incorporation of caged monomers during de novo chemical or enzymatic assembly of biopolymers (typically for oligonucleotides and oligopeptides). For proteins, systems for biosynthetic incorporation of photocaged lysine, tyrosine, serine, cysteine, and selenocysteine residues have been described (Riggsbee and Deiters, 2010Riggsbee C.W. Deiters A. Recent advances in the photochemical control of protein function.Trends Biotechnol. 2010; 28: 468-475Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In particular, a high nucleophilicity of the thiol moiety in cysteine residues has long been used for installing a wide variety of functionalities (Chalker et al., 2009Chalker J.M. Bernardes G.J.L. Lin Y.A. Davis B.G. Chemical modification of proteins at cysteine: opportunities in chemistry and biology.Chem. Asian J. 2009; 4: 630-640Crossref PubMed Scopus (426) Google Scholar). Cys has a relatively low natural abundance, and solvent-exposed free cysteines are rather uncommon in wild-type proteins. An even more unique and powerful functionality in proteins is endowed by the 21st amino acid, selenocysteine (Arnér, 2010Arnér E.S. Selenoproteins-What unique properties can arise with selenocysteine in place of cysteine?.Exp. Cell Res. 2010; 316: 1296-1303Crossref PubMed Scopus (202) Google Scholar). A few strategies for efficient biosynthesis of proteins containing photocaged Cys or Sec residues at genetically predetermined positions have recently been proposed (Lemke et al., 2007Lemke E.A. Summerer D. Geierstanger B.H. Brittain S.M. Schultz P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid.Nat. Chem. Biol. 2007; 3: 769-772Crossref PubMed Scopus (164) Google Scholar; Nguyen et al., 2014Nguyen D.P. Mahesh M. Elsässer S.J. Hancock S.M. Uttamapinant C. Chin J.W. Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells.J. Am. Chem. Soc. 2014; 136: 2240-2243Crossref PubMed Scopus (99) Google Scholar; Peeler et al., 2020Peeler J.C. Falco J.A. Kelemen R.E. Abo M. Chartier B.V. Edinger L.C. Chen J. Chatterjee A. Weerapana E. Generation of recombinant mammalian selenoproteins through genetic code expansion with photocaged selenocysteine.ACS Chem. Biol. 2020; 15: 1535-1540Crossref PubMed Scopus (10) Google Scholar; Rakauskaitė et al., 2015Rakauskaitė R. Urbanavičiūtė G. Rukšėnaitė A. Liutkevičiūtė Z. Juškėnas R. Masevičius V. Klimašauskas S. Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines.Chem. Commun. 2015; 51: 8245-8248Crossref PubMed Google Scholar; Uprety et al., 2014Uprety R. Luo J. Liu J. Naro Y. Samanta S. Deiters A. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.Chembiochem. 2014; 15: 1793-1799Crossref PubMed Scopus (38) Google Scholar). The 4,5-dimethoxy-2-nitrobenzyl (DMNB) caging group shields highly reactive Sec (or solvent exposed Cys) from undesired side reactions inside producing cells and during subsequent manipulations and can be readily removed by illumination with 365 nm light (Shao and Xing, 2010Shao Q. Xing B. Photoactive molecules for applications in molecular imaging and cell biology.Chem. Soc. Rev. 2010; 39: 2835-2846Crossref PubMed Scopus (110) Google Scholar). However, isolation of DMNB-caged proteins or their manipulations in vitro or inside cells requires additional tags or the development of individual protocols for each new protein since no affinity ligands are known to specifically interact with this relatively small moiety. To expand the general applicability and functional capability of this technology, we went on to develop a non-covalent binder that is specific toward this small chemical group (Figure 1). This was achieved by raising a monoclonal antibody against a chemically DMNB-modified carrier protein in mice. Detailed characterization of the produced selective binder demonstrated its utility for selective analysis and targeted manipulation of chemically and biosynthetically DMNB-caged proteins. The DMNB group falls in a category of very small haptens (<300 Da), which are incapable of inducing a sufficient immune response required for antibody production (Chappey et al., 1994Chappey O. Debray M. Niel E. Scherrmann J.M. Association constants of monoclonal antibodies for hapten: heterogeneity of frequency distribution and possible relationship with hapten molecular weight.J. Immunol. Methods. 1994; 172: 219-225Crossref PubMed Scopus (41) Google Scholar). To increase the immunogenicity, the hapten was covalently coupled to the KLH carrier protein, a well-established immunogen. KLH-DMNB conjugates were produced by direct chemical derivatization of accessible (predominantly) Cys residues with DMNB bromide. Alternatively, to increase the number of reactive –SH groups, KLH was first treated with a sulfhydryl-addition reagent, SATA (N-succinimidyl-S-acetylthioacetate), deacylated with hydroxylamine and then conjugated with DMNB resulting in a KLH-AT-DMNB conjugate (Figure S1). This conjugate was used to immunize mice, whereas similarly prepared BSA-DMNB and BSA-AT-DMNB conjugates were used to evaluate the specificity of the antibodies. The BSA-DMNB and BSA-AT-DMNB conjugates contained a mixture of proteins carrying 1–3 DMNB or 5–12 ATA groups (determined after first reaction with SATA), respectively (Figures S2 and S3). Hybridoma cell lines for anti-DMNB monoclonal antibody production were created using BALB/c mice. A total of eight DMNB-positive clones were selected, multiplied, and stabilized by recloning. All hybridomas produced MAbs of IgG class (Table S1). The specificity and affinity of the antibodies was evaluated by an ELISA immunoassay using the immunization antigen KLH-AT-DMNB as well as unrelated conjugates BSA-DMNB and BSA-AT-DMNB (Table S1; see Table S2 for derivatized proteins details). Of eight selected hybridomas, seven produced DMNB-specific MAbs that interacted only with DMNB-proteins but not with the carrier proteins (Figure S4). The apparent dissociation constants of DMNB-specific MAbs were in the low nanomolar to picomolar range indicating a high affinity toward the antigens. We selected the best behaved monoclonal antibody of IgG2a subclass (clone 12B6 in Figure S4, further referred to as Photo Cage Selective Binder, PCSB), which afforded excellent interaction with bacterial Protein A and thus permitted facile coupling to accessory components such as proteins or solid particles (Figure 1). Robust interactions of PCSB observed in immunoassays (Figure S5) were further confirmed by western blot analyses. PCSB not only bound KLH and BSA proteins carrying multiple DMNB groups but also was sensitive at more stringent conditions—identifying the biosynthetic EGFP Y39C-DMNB protein (see Table S3 for recombinant constructs details) labeled with a unique DMNB group (Figure 2A). Remarkably, in these experiments PCSB was able to discriminate against a chemically similar 4,5-(methylenedioxy)-2-nitrophenyl)ethyl (MDNPE) group (Figures 2A and 2C). Further western blot analyses revealed that PCSB interacted with the antigen irrespective of whether the DMNB protecting group was attached to a S or Se atom in EGFP Y39C-DMNB and EGFP Y39U-DMNB proteins, respectively. These interactions were lost when DMNB groups were removed from the proteins upon 365 nm light exposure (Figure 2B). Using western blotting of serial dilutions of purified proteins, we determined the detection limits to be 16 ng for BSA-(DMNB)1-3, 31 ng for EGFP N150C-DMNB, and 63 ng for SUMOstar E89C-DMNB (Figure S6). Altogether, these data indicate that the generated PCSB performed as a highly sensitive and selective tool suitable for DMNB group recognition. To explore the capacity of PCSB to detect DMNB-caged proteins in complex biological mixtures, we expanded the repertoire of model proteins by including three well-studied bacterial DNA cytosine-5 methyltransferases: M.HhaI, M.HpaII, and M2.Eco31I. These medium-sized proteins use their essential catalytic Cys residues to facilitate the transmethylation of target cytosine residues and can be engineered to deposit larger chemical moieties to DNA (Lukinavičius et al., 2012Lukinavičius G. Lapinaitė A. Urbanavičiūtė G. Gerasimaitė R. Klimašauskas S. Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.Nucleic Acids Res. 2012; 40: 11594-11602Crossref PubMed Scopus (36) Google Scholar). Biosynthetic expression of these recombinant proteins, produced in a photocaged and thus catalytically mute form (SUMOstar-M.HhaI C81C-DMNB, SUMOstar-M.HpaII C103C-DMNB, SUMOstar-M2.Eco31I C232C-DMNB), was monitored by western blot analysis of total yeast cell lysates (Figure S7). As expected, DMNB-positive blot signals were observed only in samples containing the expressed recombinant proteins carrying the DMNB-Cys residue but showed no interaction with controls expressing non-caged protein variants (containing Leu or Ile residues) or samples prepared from uninduced cells. Next, we checked if the interaction between the small DMNB group and the PCSB was sufficiently high to allow manipulations of native proteins by immunoprecipitation (IP) in solution. For this purpose, we first used chemically labeled DMNB-proteins carrying multiple DMNB groups to form immunoprecipitation complexes on Sepharose-Protein A beads preincubated with PCSB. The composition of the resulting IP complexes was revealed by western blot analysis of the denatured extractions of IP suspensions fractionated using gel electrophoresis. Both model methyltransferases M.HpaII-(DMNB)1-5 and M2.Eco31I-(DMNB)≥2 proteins were equally well immunoprecipitated from their pure solutions as well as from complex molecular mixtures that were prepared by adding chemically labeled DMNB-protein preparation to a total yeast cell lysate (Figure S8). Control IP reactions indicated that unlabeled recombinant proteins, components of yeast cell lysate, or unrelated isotypic MAbs showed no interference during IP. Similarly, several mutant variants of chemically labeled EGFP-DMNB and SUMOstar-DMNB proteins were found to be able to form the IP complexes (Figure S9). Further, we performed IP experiments using a series of biosynthetic recombinant protein variants each caged with a single DMNB group incorporated at a genetically defined position. Three of five analyzed EGFP-DMNB protein variants and three of four SUMOstar-DMNB protein variants showed a high or very high IP efficiency (Figures 3A and 3B ). Moreover, we were able to isolate the expressed SUMOstar I106DMNB-C protein out of the yeast cell lysate (Figure 3C). These data indicate that a single DMNB group sufficiently exposed on a surface of a native protein can be used for manipulating the latter using the generated PCSB. A brief irradiation with UV light leads to photolysis of the DMNB caging group releasing a cage-free target protein from the IP complex. A light controlled release of a chemically caged EGFP D117C(DMNB)1-3 protein from an IP complex was observed as reduction of fluorescence intensity on beads and simultaneous increase of fluorescence intensity in solution (Figures 4A–4C ). SDS-PAGE, HPLC-ESI/MS, and western blot analysis of the reaction showed the expected size of the EGFP D117C protein in solution (Figures 4D and 4E). Analogous results were obtained with a chemically caged SUMOstar I106C-(DMNB)1-2 protein (Figure S10) and biosynthetic single-label SUMOstar I106C-DMNB protein extracted from a yeast cell lysate (Figures 4D and 4F). In the latter case, Ni++ affinity chromatography of biosynthetic SUMOstar I106C-DMNB from yeast cells showed that a prevailing fraction of the produced protein was truncated at the antepenultimate position (SUMOstar105ter, residues 1–105) (Figure 4F, top panel). Remarkably, during the IP procedure, only the full length protein (residues 1–108) has been effectively pulled out from a dilute cell lysate and photolytically recovered in a pure label-free form with no detectable amounts of the contaminating SUMOStar105ter variant (Figure 4F, bottom panel). Here we report the development of the first highly selective non-covalent binder for a popular photocaging group that can be biosynthetically incorporated into recombinant proteins in yeast (Lemke et al., 2007Lemke E.A. Summerer D. Geierstanger B.H. Brittain S.M. Schultz P.G. Control of protein phosphorylation with a genetically encoded photocaged amino acid.Nat. Chem. Biol. 2007; 3: 769-772Crossref PubMed Scopus (164) Google Scholar; Rakauskaitė et al., 2015Rakauskaitė R. Urbanavičiūtė G. Rukšėnaitė A. Liutkevičiūtė Z. Juškėnas R. Masevičius V. Klimašauskas S. Biosynthetic selenoproteins with genetically-encoded photocaged selenocysteines.Chem. Commun. 2015; 51: 8245-8248Crossref PubMed Google Scholar) and mammalian (Nguyen et al., 2014Nguyen D.P. Mahesh M. Elsässer S.J. Hancock S.M. Uttamapinant C. Chin J.W. Genetic encoding of photocaged cysteine allows photoactivation of TEV protease in live mammalian cells.J. Am. Chem. Soc. 2014; 136: 2240-2243Crossref PubMed Scopus (99) Google Scholar; Peeler et al., 2020Peeler J.C. Falco J.A. Kelemen R.E. Abo M. Chartier B.V. Edinger L.C. Chen J. Chatterjee A. Weerapana E. Generation of recombinant mammalian selenoproteins through genetic code expansion with photocaged selenocysteine.ACS Chem. Biol. 2020; 15: 1535-1540Crossref PubMed Scopus (10) Google Scholar) cells or installed de novo by chemical or chemoenzymatic labeling (Anhäuser et al., 2018Anhäuser L. Muttach F. Rentmeister A. Reversible modification of DNA by methyltransferase-catalyzed transfer and light-triggered removal of photo-caging groups.Chem. Commun. (Camb.). 2018; 54: 449-451Crossref PubMed Google Scholar; Heimes et al., 2018Heimes M. Kolmar L. Brieke C. Efficient cosubstrate enzyme pairs for sequence-specific methyltransferase-directed photolabile caging of DNA.Chem. Commun. 2018; 54: 12718-12721Crossref PubMed Google Scholar). This new tool effectively expands the functionality of the photocaging moiety into a useful orthogonal affinity handle. Despite the small size of the target group, the present PCSB displays a high affinity (nM–pM) and specificity toward a range of DMNB-caged proteins and permits selective determination of nanogram quantities of target proteins or analysis of DMNB-proteomes in biological milleux by western blotting. Consistent with the relative bulkiness of the antibody Fab region (Chiu et al., 2019Chiu M.L. Goulet D.R. Teplyakov A. Gilliland G.L. Antibody structure and function: the basis for engineering therapeutics.Antibodies. 2019; 8: 55Crossref Google Scholar), we found that binding of the DMNB group in native proteins was strongly dependent on its accessibility. However, for both model systems examined, multiple surface positions were identified that permitted selective PCSB capture of biosynthetically produced or chemically modified proteins (Figure 3). In light of a growing interest in optogenetics (Guglielmi et al., 2016Guglielmi G. Falk H.J. De Renzis S. Optogenetic control of protein function: from intracellular processes to tissue morphogenesis.Trends Cell Biol. 2016; 26: 864-874Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and nucleic acids-based nanodevices (Chakraborty et al., 2016Chakraborty K. Veetil A.T. Jaffrey S.R. Krishnan Y. Nucleic acid–based nanodevices in biological imaging.Annu. Rev. Biochem. 2016; 85: 349-373Crossref PubMed Scopus (87) Google Scholar), it will be attractive to determine the PCSB accessibility of DMNB groups located in single-stranded oligonucleotides and double stranded DNA/RNA structures. The presented series of examples demonstrates the first general approach in which a selective capture and traceless release of a caged protein can be achieved in a light-controlled fashion under near physiological non-denaturating conditions (Figures 4 and S10). Such tools are highly desirable for laboratory production of designer proteins, or even less accessible selenoproteins, and expand the availability and diversity of functionalized biomolecules for applications in miniature light-controlled systems involving microfluidic devises, microchips, and nanoparticles (Yang et al., 2017Yang Y. Mu J. Xing B. Photoactivated drug delivery and bioimaging.Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2017; 9: e1408Crossref Scopus (48) Google Scholar; Young and Schultz, 2018Young D.D. Schultz P.G. Playing with the molecules of life.ACS Chem. Biol. 2018; 13: 854-870Crossref PubMed Scopus (149) Google Scholar; Khamo et al., 2017Khamo J.S. Krishnamurthy V.V. Sharum S.R. Mondal P. Zhang K. Applications of optobiology in intact cells and multicellular organisms.J. Mol. Biol. 2017; 429: 2999-3017Crossref PubMed Scopus (22) Google Scholar; Silva et al., 2019Silva J.M. Silva E. Reis R.L. Light-triggered release of photocaged therapeutics - where are we now?.J. Control. Release. 2019; 298: 154-176Crossref PubMed Scopus (59) Google Scholar; Sortino, 2012Sortino S. Photoactivated nanomaterials for biomedical release applications.J. Mater. Chem. 2012; 22: 301-318Crossref Google Scholar). Immunocapture of native proteins is limited to those that carry surface-exposed DMNB groups. On the other hand, PCSB interactions with SDS-denatured proteins during western blot analyses proved effective independently of their tag location. Photochemical decaging using UV light is known to generate free radicals and reactive oxygen species, which may be detrimental to native proteins and other macromolecules Kerwin and Remmele, 2007Kerwin B.A. Remmele Jr., R.L. Protect from light: photodegradation and protein biologics.J. Pharm. Sci. 2007; 96: 1468-1479https://doi.org/10.1002/jps.20815Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar. Therefore, inclusion of radical/oxygen scavenging compounds such as methionine in reaction buffers and use of inorganic material-based solid particles for assembly of IP complexes is advised. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Saulius Klimašauskas ( [email protected] ). Plasmids generated in this study will be made available upon request. The antibody generated in this study will be made available on request, but we may require a payment and/or a completed Materials Transfer Agreement. This study did not generate datasets or codes. All methods can be found in the accompanying Transparent Methods supplemental file. The authors thank Audronė Rukšėnaitė for HPLC/ESI-MS data acquisition, Žilvinas Kožemekinas for assistance with preparation of MNDPE-Cys and Pranciškus Vitta for technical support with the LED light source. This work was supported by the Research Council of Lithuania (grant S-MIP-17-57 to S.K.) and Vilnius University Infrastructure Support Fund. Conceptualization, S.K. and A.Ž.; Methodology, Investigation, and Validation, R.R., G.U., M.S., R.L., and A.V.; Resources, G.P. and V.M.; Data Curation, G.U.; Writing – Original draft, R.R., G.U., M.S., V.M., and S.K.; Writing – Review & Editing, R.R. and S.K; Supervision, S.K., A.Ž., and V.M.; Project Administration, R.R. and S.K.; Funding Acquisition, S.K. R.R., G.U., M.S., R.L., A.Ž., and S.K. are inventors on a related patent application. Download .pdf (2.07 MB) Help with pdf files Document S1. Transparent Methods, Figures S1–S10, and Tables S1–S3" @default.
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• W3107524262 title "Photocage-Selective Capture and Light-Controlled Release of Target Proteins" @default.
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