DOI: 10.1002/open.201900038 Communications 1 2 3 Efficient bioactive oligonucleotide-protein conjugation for 4 5 cell-targeted cancer therapy 6 7 Anna Aviñó,*[a, b] Ugutz Unzueta,[b, c] María Virtudes Céspedes,[b, c] Isolda Casanova,[b, c] 8 Esther Vázquez,[b, d, e] Antonio Villaverde,[b, d, e] Ramon Mangues,[b, c] and Ramon Eritja*[a, b] 9 10 Oligonucleotide-protein conjugates have important applications end, DNA-protein conjugates have been used to fabricate 11 in biomedicine. Simple and efficient methods are described for arrangements of proteins using the specificity of base-pairing of 12 the preparation of these conjugates. Specifically, we describe a nucleic acids or to immobilize proteins to surfaces in the 13 new method in which a bifunctional linker is attached to thiol- fabrication of protein arrays or other supramolecular devices.[3–5] 14 oligonucleotide to generate a reactive intermediate that is used Additionally, several immunological bioassays are based on the 15 to link to the protein. Having similar conjugation efficacy use of these chimeras.[6] 16 compared with the classical method in which the bifunctional The attachment of oligonucleotides to proteins or other 17 linker is attached first to the protein, this new approach polymers have shown to enhance nucleic acids delivery in gene 18 produces significantly more active conjugates with higher batch inhibition strategies.[7,8] In this sense, polymer-DNA drug con- 19 to batch reproducibility. In a second approach, direct conjuga- jugates are one of the approaches to modify solubility, bio- 20 tion is proposed using oligonucleotides carrying carboxyl logical activity and stability of these drugs.[9] 21 groups. These methodologies have been applied to prepare Several methods have been proposed to link synthetic 22 nanoconjugates of an engineered nanoparticle protein carrying oligonucleotides to proteins using both non-covalent and 23 a T22 peptide with affinity for the CXCR4 chemokine receptor covalent approaches. The reversible non-covalent strategy (e. g. 24 and oligomers of the antiproliferative nucleotide 2’-deoxy-5- biotin-streptavidin[10] or nickel-histidine[11]) produce poorly sta- 25 fluorouridine in a very efficient way. The protocols have ble conjugates with difficult handling.[7,12] A wide variety of 26 potential uses for the functionalization of proteins, amino- chemistries for covalent conjugation have been reported.[1–4] 27 containing polymers or amino-lipids in order to produce Some of them are based on the modification at a predeter- 28 complex therapeutic nucleic acid delivery systems. mined site of the oligonucleotides with reactive groups that 29 directly react with the amino groups of lysines or the thiol 30 groups of cysteines obtaining amide or disulfide bonds 31 respectively.[13,14] More complex strategies have also been 32 Oligonucleotide-protein conjugates hold great promise for reported using post-transcriptional modification of the protein 33 studying DNA-protein interactions while being versatile molec- or involvement of a catalytic or reactive protein domain.[15] 34 ular tools for biotechnology and material science.[1–3] To this Since biomolecules are often present at low concentrations, the 35 conjugation depends on the reaction rate and the stability of 36 [a] Dr. A. Aviñó,+ Prof. Dr. R. Eritja the reacting groups. More efficient results were obtained using 37 Institute for Advanced Chemistry of Catalonia (IQAC) bioorthogonal approaches in which the reactive groups do not 38 Spanish Council for Scientific Research (CSIC) modify the activity of the biomolecules. Specifically, bioorthog- 39 Jordi Girona 18–26, 08034 Barcelona, Spain E-mail: aaagma@cid.csic.es onal methods such as copper-catalyzed azide-alkyne [3 + 2] 40 recgma@cid.csic.es cycloaddition[12] or oxime and hydrazone ligation have been 41 [b] Dr. A. Aviñó,+ Dr. U. Unzueta,+ Dr. M. Virtudes Céspedes, Dr. I. Casanova, proposed.[16] In addition, several bifunctional cross-linkers have 42 Dr. E. Vázquez, Prof. Dr. A. Villaverde, Prof. Dr. R. Mangues, Prof. Dr. R. Eritja Networking Center on Bioengineering, Biomaterials and Nanomedicine been also described to produce protein conjugates.[17,18] The 43 (CIBER-BBN) most frequently used bifunctional linkers carry maleimide and 44 [c] Dr. U. Unzueta,+ Dr. M. Virtudes Céspedes, Dr. I. Casanova, N-hydroxysuccinimidyl ester groups. These compounds are first 45 Prof. Dr. R. Mangues Institut d’Investigacions Biomèdiques Sant Pau and Josep Carreras Resarch coupled with the protein to produce maleimide-protein inter- 46 Institute, Hospital de Santa Creu i Sant Pau, 08025 Barcelona, Spain mediates. Then, after removal of the excess of cross-linker, the 47 [d] Dr. E. Vázquez, Prof. Dr. A. Villaverde maleimide group is reacted with a thiol modified oligonucleo- 48 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain tide. However, inversion of the reactive groups has also been 49 [e] Dr. E. Vázquez, Prof. Dr. A. Villaverde described in which amino modified DNA reacts with the 50 Departament de Genètica i de Microbiologia, Universitat Autònoma de biofunctional linker producing a maleimide-oligonucleotide 51 Barcelona, 08193 Bellaterra, Spain that finally reacts with the free thiol of cysteines of the 52 Supporting information for this article is available on the WWW under https://doi.org/10.1002/open.201900038 protein.[19] 53 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. Multifunctional proteins have emerged as powerful engi- 54 This is an open access article under the terms of the Creative Commons neered chimeric proteins that incorporate several selected 55 Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited domains. Among them, protein-based nanocarriers based on 56 and is not used for commercial purposes. the concept of virus-mimetic vehicles have been explored for 57 ChemistryOpen 2019, 8, 382 – 387 382 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Communications drug delivery.[20] Some of us have developed protein nano- treated with an excess of a bifunctional cross-linker 6- 1 particles for targeted drug delivery to tumor cells that over- Maleimidohexanoic acid N-hydroxysuccinimide ester (EMCS) 2 express the chemokine receptor CXCR4. This receptor is and the resulting maleimide functionalized protein is reacted 3 associated with metastatic progression in several tumor types, with the 3’-thiol-oligonucleotide. This two-step protocol was 4 including colorectal cancer and in human immunodeficiency efficient but time-consuming as the excess of the EMCS has to 5 virus infection.[21–23] This carrier incorporates the T22 peptide, a be removed from the protein by dialysis and the maleimide 6 known ligand of CXCR4, which internalizes into CXCR4 + cells group attached to the protein can slowly react with amino 7 via specific receptor endocytosis.[24,25] This engineered self- groups of Lys that contains the protein. This really was 8 assembling protein incorporates a green fluorescent protein inconvenient because we observed in some cases a low 9 and a histidine tag.[26] The resulting T22-GFP-H6 protein nano- functionalization of the protein due to the decrease of available 10 particle has been reported to be an excellent drug carrier in cell maleimide groups to react with the 3’-thiol-oligonucleotide and 11 culture and for in vivo targeting to cells that overexpress the low batch to batch reproducibility. In order to solve these 12 CXCR4 receptor in metastatic colorectal cancer mouse problems we studied the reversal of the order of the reactants 13 models.[27] by reacting first the bifunctional crosslinker with the thiol- 14 5-Fluoro-2’-deoxyuridine (FdU) is a cytotoxic drug known for oligonucleotide and then the resulting oligonucleotide carrying 15 its high antitumor activity against cancer.[28] Short oligomers the N-hydroxysuccinimidyl ester was used for the functionaliza- 16 carrying FdU have been described as prodrugs producing FdU tion of the protein (Scheme 1B and supporting information S8- 17 monophosphate inside the cells.[29–32] FdU monophosphate is S10). 18 the active form of FdU as it can be converted to FdU Additionally, the formation of FdU-oligonucleotide-protein 19 triphosphate and induce the synthesis of mutated DNA that conjugates was also studied via direct amide formation using 20 provoke cell death. In addition FdU monophosphate is an FdU oligomers carrying 5’-carboxyl groups (Scheme 1C and 21 inhibitor of thymidine synthase producing thymine-less cell supporting information S10–S12). This approach avoids the use 22 death. The use of short FdU oligomers for cancer treatment has of bifunctional linkers. 23 been described in the bibliography[29,32] but delivery of the In the present communication we show that the new 24 antiproliferative oligomers remains an unsolved problem.[30,31] approaches B and C are feasible, simpler and able to produce 25 Our present interest is to functionalize this T22-GFP-H6 protein the oligonucleotide-protein conjugates with antiproliferative 26 nanoparticle with nucleic acid drugs for cancer treatment. To activity in one-pot reaction with equal or slightly higher 27 this end, we have selected the functionalization of T22-GFP-H6 efficiency. Additionally, these methods avoid the potential 28 protein with oligonucleotides carrying several units of FdU cross-linking of the protein as it was observed in the first 29 using the method based on the thiol-maleimide reaction. We method and, therefore, the conjugation rate could be kept 30 have demonstrated that this conjugate showed antitumor but constant between the different conjugate batches. However, 31 specifically antimetastatic activity in a CXCR4 + metastatic although apparently similar product is obtained by all method- 32 colorectal cancer model.[33] ologies in terms of nanoconjugate size and drug payload, 33 The classical method consists of two steps (Scheme 1A, and completely different functional performance is observed for the 34 supporting information S2-S8). First, T22-GFP-H6 protein is nanoconjugates; showing that the used conjugation method- 35 ology exerts high impact in resulting nanoconjugates function- 36 ality even though showing similar physicochemical properties. 37 T22-GFP-H6-(FdU)5 nanoconjugates were generated by 38 covalent binding of the T22-GFP-H6 nanoparticles and oligom- 39 ers carrying five units of FdU (5’-(FdU)5-hexaethyleneglycol- 40 thiol-3’). For comparison purposes the classical approach 41 (Scheme 1A) was also performed (see supporting information, 42 S2–S8). The optimal conditions for the classical two-step 43 approach are the use of 5 molar excess of the linker with 44 respect to the protein followed by an overnight treatment with 45 5 molar excess of thiol-oligonucleotide. These conditions 46 generate conjugates with an average number of around 3 47 oligonucleotides per protein that corresponds to 15 molecules 48 of the FdU drug (Table 1). These oligonucleotide/protein ratios 49 Scheme 1. Representation of the reactions involved in the preparation of are considered optimal without compromising the functionality 50 oligonucleotide-FdU protein conjugates. (A) The classical method involving of the protein (binding to the CXCR4 receptor and 51 the addition of the maleimide group to the protein and subsequent Michael addition of thiol-oligonucleotide. (B) Inverted one-pot addition method in internalization).[33] Unfortunately, using this protocol, we ob- 52 which the 3’-thiol-oligonucleotide reacts with the maleimide group of the served batch to batch differences in terms of FdU protein 53 linker and then the oligonucleotide carrying the N-hydroxysuccinyl ester loading. To solve this problem, we studied the excess of the 54 group reacts with the protein. (C) Direct method in which the 5-carboxy- oligonucleotide is activated and then conjugated with the protein nano- linker in the coupling reaction to the protein nanoparticle. The 55 particle. Nanoparticle in-silico representation modified from (34). Reprinted results, confirmed by gel electrophoresis, indicate that when we 56 by permission of John Wiley & Sons, Inc. increase the linker excess, more bands corresponding to cross- 57 ChemistryOpen 2019, 8, 382 – 387 www.chemistryopen.org 383 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Communications 1 Table 1. Functionalization, size and antiproliferative activity (MTT assay) of the protein conjugates carrying FdU oligomers prepared in this work. 2 Conjugates Method Conditions Units of oligo-drug Size by DLS MTT (IC50) HeLa 3 /proteina 4 T22-GFP-H6-FdU Classical two-step protocol 1 mg/ml, × 5 drug excess 3 FdU/protein 11–14 nm 16.22 nM 5 T22-GFP-H6-FdU Inverted one-pot addition 2 mg/ml, × 5 drug excess 3 FdU/protein 11–12 nm 2.08 nM T22-GFP-H6-FdU Direct -COOH coupling 2 mg/ml, × 5 drug excess 1.2 FdU/protein 9.5–13 nm 2.84 nM 6 BSA-FdU Inverted one-pot addition 2 mg/ml, x 5 drug excess 3 FdU/protein 7.4 nm n.d. 7 T22-GFP-H6-FdU Inverted one-pot addition 2 mg/ml, × 50 drug excess 5.9 FdU/protein 11–12 nm 0.43 nM 8 T22-GFP-H6-FdU Direct -COOH coupling 2 mg/ml, × 50 drug excess 4.9 FdU/protein 9.5–13 nm 1.08 nM 9 n.d.: not determined. aAverage number of oligonucleotide units per protein measured by UV spectroscopy 10 11 12 linked nanoparticles trapped into the gel well are observed. reaction the yields are good if one considers that using a 5 13 Consequently, fewer operative nanoparticles are obtained that times excess of thiol-oligonucleotide, approx. 3 molecules of 14 may affect the subsequent FdU loading (supporting information oligonucleotide are attached to the protein. This suggests that 15 Figure S5). The potential side reaction of the amino groups of the thiol group reacted mainly with the maleimide group of the 16 lysines to maleimido groups is in agreement with the observed bifunctional linker although we cannot discard some side 17 instability of maleimido lysine-rich peptides described by Eritja reaction between thiol group and the active ester to yield a 18 et al.[35] thioester oligonucleotide dimer that slowly hydrolyze to the 19 Based on the selective reactivity of thiol-oligonucleotides oligonucleotide-carboxylic acid. 20 for the maleimido function[35] we decided to study the reversal The inverted one-pot method was extended to produce 21 of the order of the reactants (scheme 1B). In a first trial the conjugates with larger proteins. We choose BSA (Bovine Serum 22 thiol-oligonucleotide was mixed with ECMS in a 1 : 1 molar ratio Albumin Fraction V, Roche). BSA has a molecular weight of ~ 68 23 and after 10 min of incubation time at room temperature the KDa being twice the size the monomeric T22-GFP-H6 protein. 24 reaction mixture was added to the protein T22-GFP-H6 protein BSA has 59 lysine residues, of which 30–35 have exposed 25 obtaining the desired protein-oligonucleotide conjugate. To our primary amines that can react with the bifunctional cross-linker. 26 knowledge the selective reaction of thiol-oligonucleotides to For comparison, T22-GFP-H6 contains 23 Lys residues, all of 27 maleimido groups in the presence of the N-hydroxysuccinimidyl them exposed. We have used the optimized inverted addition 28 ester has not been reported. Next, we optimized the one-pot protocol to produce BSA-(FdU)5 conjugates obtaining an 29 inverted addition method in which the thiol-oligonucleotide is average of 3 molecules of oligo-(FdU)5 per protein (supporting 30 reacted first with equimolar amounts of the EMCS linker (for information, S9, S10). It is worth to mention that in the case of 31 10 min at room temperature) and then the mixture was added BSA in spite of being a bigger protein than T22-GFP-H6 the DLS 32 over the T22-GFP-H6 nanoparticles solution at a 1 : 5 (nano- data indicate a smaller size (7.4 nm) compared with T22-GFP- 33 particles: oligo-FdU) molar ratio for 2 h at room temperature H6. This is because T22-GFP-H6 self-assemble to produce a 34 followed by an overnight incubation at 4 °C. The second step larger protein nanoparticle.[33,34] 35 implies the amide formation by the activated ester of the linker The key step in the formation of the conjugates is the 36 functionalized oligo-FdU and the amino groups of the external production of pure, active and sufficient activated products to 37 Lys of the T22-GFP-H6. Following this method we obtained an conjugate with the protein. Next, we thought it was worth 38 average functionalization of up to 3 molecules of oligo-FdU per preparing an activated oligo-FdU without using bifunctional 39 protein depending on the conditions, which corresponds to 15 linkers. For that, conjugates were also obtained via direct 40 molecules of FdU (see supporting information, S8–S10). coupling of activated COOH-oligo-FdU and the protein nano- 41 Although the efficiency is similar using both methods, the particle (Scheme 1C). A carboxylic group was added to the 5’- 42 second is faster as no dialysis is required to remove the linker end of the oligo-FdU, then it was activated with sulfo-NHS (N- 43 from the oligo-FdU. Most importantly, we did not observe any hydroxysulfosuccinimide) and EDC (N-(3-Dimethylaminopropyl)- 44 cross-linking between different proteins (Figure S10) as we saw N’-ethylcarbodiimide) and, finally, it was allowed to react with 45 in the classical method (Figure S5) even at higher concentration the protein. Moderate efficacy (1.2 oligo-FdU) was obtained 46 of the linker or the protein (2 mg/ml), which is probably due to using the 5–10 molar excess (see supporting information, S10– 47 the reaction of lysines to a maleimido group. The best results in S12). 48 the one-pot inverted method were obtained performing an Optimization of the reaction was accomplished using 50- 49 overnight conjugation at room temperature and at 2 mg/ml fold excess of activated oligonucleotide followed by an over- 50 protein concentration (Table 1). The analysis by mass spectrom- night reaction with the protein at 2 mg/ml. Using these 51 etry of the dialyzed solutions after the coupling of the thiol- conditions we obtained 4.9 FdU oligonucleotide units per 52 oligonucleotide showed the presence of the oligonucleotide- protein nanoparticle using the direct method (see supporting 53 carboxylic acid coming from the hydrolysis of the N-hydrox- information). Similarly, using the same conditions, in the 54 ysuccinimidyl ester. This compound was expected because of inverted one-pot addition, we obtained 5.9 FdU oligonucleotide 55 the use of aqueous media. Even with the potential hydrolysis of units that was the highest ratio of oligo-FdU coupled to the 56 the N-hydroxysuccinimide ester during the thiol-maleimido nanoparticle protein. T22-GFP-H6-FdU conjugates were used for 57 ChemistryOpen 2019, 8, 382 – 387 www.chemistryopen.org 384 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Communications blocking CXCR4 + metastatic colon cancer stem-cells.[33] Using 1 these conditions, concentrated conjugates were obtained that 2 were highly recommended for clinical testing as we reduced 3 the volume required for intravenous administration. 4 Mass spectrometry of all conjugates confirmed the addition 5 of oligo-FdU to the proteins (supporting information, Figur- 6 es S2, S6-S9). Figure 1A shows the spectrum of T22-GFP-H6 7 nanoparticle and T22-GFP-H6-FdU conjugate prepared using 8 inverted one-pot addition. 9 The volume size distribution of T22-GFP-H6 nanoparticles 10 and T22-GFP-H6-FdU conjugates was also measured by Dynam- 11 ic Light Scattering (Table 1 and Figure 1B). No significant 12 particle size differences were observed. 13 Antiproliferative activity of FdU-protein conjugates was 14 measured by MTT assays in CXCR4 overexpressing (CXCR4 +) 15 HeLa cells (see Figure 1C and supporting information S5). The Figure 2. Functional characterization of nanoconjugates. T22-GFP-H6-FdU 16 payload-response trend line representation. Antitumor IC50 concentration of more active FdU conjugate was the T22-GFP-H6-FdU nano- 17 each sample was calculated by dose-response curves in MTT cell viability particle conjugate prepared by inverted one-pot method and is assays upon exposure over CXCR4 + HeLa cells for 48 h. Grey dots represent 18 in accordance with the higher FdU loading (Table 1). At 5 molar samples generated by inverted one-pot addition, blue dots represent 19 samples generated by direct-COOH coupling and purple dots represent excess of drug, the conjugate prepared by inverted one-pot 20 samples generated by classical two-step protocol. Note that sample from addition has 8 times higher antiproliferative activity than the classical two-step protocol doesn’t fit in linear regression line. 21 classical two-step protocol, although both have the same FdU 22 loading. This indicates that the new method is more efficient. In 23 the direct method, the antiproliferative effect is more than 5 24 times higher than the classic method, although only 1.2 of lineal pattern as the inverted method. In both methods, the IC50 25 oligo-FdU is conjugated. In contrast, direct exposure of HeLa is directly proportional to the oligo-FdU conjugated, generating 26 cells to unconjugated oligo-FdU dramatically reduces the nanoconjugates with similar properties (Figure 2). However, in 27 observed antitumor activity (IC50 = 275 nM, Figure 1H, reference the classical method, the inhibitory properties not only depend 28 [33]) since this molecule could not be concentrated in the cell on the FdU payload but also other potential factors such as the 29 cytosol because it does not use receptor-mediated endocytosis proportion of the cross-linking generated during the prepara- 30 to enter the cell. tion of the nanoconjugate that may negatively affect their 31 When we increased the excess of the oligo-FdU, the functionality. 32 antiproliferative activity is slightly better in inverted one-pot We have extended this knowledge to the preparation of 33 addition method rather than the direct COOH coupling because conjugates carrying oligonucleotide-poly-L-Lysine conjugates 34 more oligo-FdU is conjugated to the nanoparticle protein. and oligonucleotide-lipid conjugate (Scheme 2, supporting 35 Likewise, dose-response viability of the conjugate T22-GFP-H6- information S12–S17). Poly-L-Lys is a hydrophilic cationic 36 FdU using direct method was similar and follows the same molecule with a high number of potential amino reacting 37 groups.[36] We employed poly-Lys, average molecular weight 38 4200 with around 20–35 available Lys residues. Octylamine was 39 used as a lipid molecule and contains a single amine. 40 The conjugation of oligonucleotides to cationic polymers 41 such as poly(L-Lys) has been described to enhance oligonucleo- 42 tide delivery to cells,[37] to stabilize duplex and triplex 43 structures[38] and to allow the introduction of multiple labels.[39] 44 However, there are few strategies to prepare poly-L-Lys- 45 conjugated oligonucleotides.[37–40] These methods require sev- 46 eral steps and are time-consuming procedures. Here we have 47 48 49 50 51 52 Figure 1. A). Mass spectrometry of the initial T22-GFP-H6 protein (in red) and 53 the T22-GFP-H6-FdU conjugate (in blue). B) DLS of the T22-GFP-H6 protein 54 (in green) and the T22-GFP-H6-FdU conjugate (in red) using inverted one- 55 pot reaction. C) Dose-response representation of CXCR4 + HeLa cells Scheme 2. Schematic representation for the preparation of lipid-oligo-FdU exposed to different concentration of T22-GFP-H6-FdU conjugates for 48 h, conjugates and polymer-oligoFdU conjugates using the inverted addition 56 analyzed by MTT viability assay. method. 57 ChemistryOpen 2019, 8, 382 – 387 www.chemistryopen.org 385 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Communications prepared the T5-thiol and (FdU)5-hexaethyleneglycol-thiol-3’ provided the expected conjugates in a reproducible and 1 and applied the one-pot inverted addition of the bifunctional efficient way obtaining similar drug/protein ratios. Importantly, 2 linker to generate poly-(L-Lys)-oligonucleotide conjugates. Two we have demonstrated that not only the number but also the 3 different conjugates were obtained, namely T5-poly-L-Lys and distribution of the drug is important in the efficacy of the 4 (FdU)5-poly-L-Lys (see supporting information). The mass spec- protein conjugate. The order of the reacting bifunctional linker 5 tra analysis revealed several sets of signals corresponding to in the synthesis of the protein conjugates plays an important 6 multiple oligonucleotides attached to poly-L-Lys molecules role in the production of nanostructures. The results confirmed 7 (Figures S14-S16, Table S1). However, the major set of signals that using the inverted-addition method, although we did no 8 corresponds to the 1 : 1 oligonucleotide-poly-L-Lys conjugate. increase the number of conjugated oligo-FdU, the antiprolifer- 9 Next, we applied the protocols for the synthesis of a simple ative effect was around eight times higher compared to 10 model oligonucleotide-lipid conjugate.[41] Lipids are also inter- classical method. In addition, we observed a higher reproduci- 11 esting molecules used to improve the delivery of the bility of the different batches of the inverted-addition compared 12 oligonucleotides. Here we tested our inverted addition method- to classical method. Similar results were obtained using the 13 ology for the addition of n-octylamine, a hydrophobic molecule direct method without using bifunctional linkers. Both ap- 14 carrying one single amino group. In this case, we obtained the proaches, inverted one-pot and direct methods, produce nano- 15 corresponding T5-octyl and (FU)5-octyl conjugates in good yield. particle conjugates with antiproliferative properties directly 16 The simplicity of the conjugates allowed the standard reversed- proportional to the FdU payload. However, the classical 17 phase HPLC purification (Figure S21) and the major peak was method, although similar physical properties are obtained (size 18 isolated and characterized by mass spectrometry (supporting and payload), the resulting product is different regarding 19 information, Figures S18–S19). The possibility of quantification functionally and much less efficient. 20 of the final product allows us studying the impact on the yields The proposed methodologies have also been applied to 21 of small changes in the conditions used in the first step. Going generate oligonucleotide conjugates with lipids or poly-L-Lys, 22 from 10 min to 30 min incubation time does not affect the with different number of amino reactive groups, proving their 23 yield, but using a 1 : 2 (thiol-DNA/EMCS) molar ratio the yield of extended applicability to different potential drug carriers. In 24 conjugate was clearly reduced (see supporting information, particular, we can envision the use of these simple and useful 25 S18–S19). This indicates that it is more critical to adjust the 1 : 1 methodologies to conjugate oligonucleotides to other mole- 26 molar ratio than the time of incubation of thiol-DNA with EMCS. cules of interest such as carbohydrates, peptides or antibodies. 27 Conjugation of biomolecules offers the possibility of 28 creating new entities with improved properties. For instance, 29 conjugation of oligonucleotides to proteins allows the assembly Acknowledgements 30 of proteins for nanostructure arrangements, biomedical diag- 31 nostics or for an efficient delivery of the therapeutic nucleic This work was supported byPlan Estatal de I + D + I 2013–201, 32 acids. Institto de Salud Carlos III and MINECO (co-funding from FEDER) 33 Bifunctional linkers, although having appeared a long time CTQ2017-84415-R, CTQ2014-52588-R, CP15/00167 and BIO2016- 34 ago, are now frequently used to generate new reacting groups 76063, AGAUR (2017SGR865, 2017SGR114 and 017SGR229), 35 for further coupling reactions. Several linkers with different MaratóTV3 (416/C/2013), andCIBER-BBN Nanomets nd Nano- 36 reacting groups, length and hydrophobicity are now commer- prother Intramural Projects. We used the CIBER-BBN Nanotoxicol- 37 cially available. Here, we explored the specific reactivity of ogy Platform and protein production and DLS have been partially 38 biofunctional linkers to generate different oligonucleotide performed by ICTS ”NANBIOSIS”, more specifically by the Protein 39 conjugates. In particular, we improved the coupling conditions Production Platform of CIBER-BBN/IBB (http://www.nanbiosis.es/ 40 compared to the classical method obtaining similar conjugates unit/u1-protein-production-platform-ppp/) and the Biomaterial 41 in less time; also, we demonstrated the versatility of the Processing and Nanostructuring Unit (http://www.nanbiosis.es/ 42 inverted one-pot addition method to modulate the number of portfolio/u6-biomaterial-processing-and-nanostructuring-unit). 43 nucleic acids derivatives attached to the proteins by changing M.V.C. and U.U. are supported by Miguel Servet contract from 44 the reaction conditions. In addition, we have demonstrated that ISCIII and by PERIS program from the health department of la 45 the direct conjugation of the biomolecules is also possible in Generalitat de Catalunya, respectively. A.V. received an ICREA 46 good yields without the use of the bifunctional linkers. This has ACADEMIA Award. Authors thank the “Servei d’ Anàlisis Química” 47 been accomplished because it is possible to introduce several of the Universitat Autònoma de Barcelona and the “Serveis 48 types of functional groups such as carboxyl groups at different Cientifico-Técnics” of the Universitat de Barcelona for their help in 49 positions of nucleic acids in a very efficient way. the analysis of the mass spectra. Nanoparticle in-silico representa- 50 In proteins, the most available reacting groups are the tion modified from (34). Reprinted by permission of John Wiley & 51 amino groups from Lys and, for this reason, are often used for Sons, Inc. 52 the conjugation with other molecules or materials. For example, 53 this approach has been used for the synthesis of the antibody- 54 drug conjugates for cancer therapies. Similarly, our methods of Conflict of Interest 55 preparing protein-nucleic acids drug conjugates rely on the 56 reactivity of the lysines. We have used coupling conditions that The authors declare no conflict of interest. 57 ChemistryOpen 2019, 8, 382 – 387 www.chemistryopen.org 386 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Communications Keywords: Oligonucleotide-protein conjugate · nanoparticle [25] T. Murakami, T.-Y. Zhang, Y. Koyanagi, Y. Tanaka, J. Kim, Y. Suzuki, S. 1 Minoguchi, H. Tamamura, M. Waki, A. Matsumoto, J. Virol. 1999, 73, protein · 2’-deoxy-5-fluorouridine · polymers · lipids 2 7489–7496. [26] U. Unzueta, M. V. Céspedes, N. Ferrer-Miralles, I. Casanova, J. Cedano, 3 J. L. Corchero, J. Domingo-Espín, A. Villaverde, R. Mangues, E. Vázquez, 4 Int. J. Nanomed. 2012, 7, 4533. [1] G. T. Hermanson, Bioconjugate techniques, Academic press, 2013. 5 [2] C. M. Niemeyer, Angew. Chem. Int. Ed. 2010, 49, 1200–1216; Angew. [27] M. V. Céspedes, U. Unzueta, P. Álamo, A. Gallardo, R. Sala, I. Casanova, Chem. 2010, 122, 1220–1238 M. A. Pavón, M. A. Mangues, M. Trías, A. Lopez-Pousa, Nanomedicine 6 [3] C. M. Niemeyer, Nano Today 2007, 2, 42–52. 2016, 12, 1987–1996. 7 [28] H. Nakagawa, N. Maeda, T. Tsuzuki, T. Suzuki, A. Hirayama, E. Miyahara, [4] C. M. Niemeyer, Trends Biotechnol. 2002, 20, 395–401. 8 [5] J. R. McMillan, J. D. Brodin, J. A. Millan, B. Lee, M. Olvera de la Cruz, C. A. K. Wada, Jpn. J. Clin. Oncol. 2001, 31, 251–258. Mirkin, J. Am. Chem. Soc. 2017, 139, 1754–1757. [29] T. S. Pardee, K. Stadelman, J. Jennings-Gee, D. L. Caudell, W. H. Gmeiner, 9 [6] F. Akter, M. Mie, E. Kobatake, Sens. Actuators B 2014, 202, 1248–1256. Oncotarget 2014, 5, 4170. 10 [30] P. Dua, S. Kim, D.-k. Lee, Nucleic Acid Ther. 2015, 25, 180–187. [7] S. Rajur, C. Roth, J. Morgan, M. Yarmush, Bioconjugate Chem. 1997, 8, 11 935—940. [31] S. Kruspe, U. Hahn, Angew. Chem. Int. Ed. 2014, 53, 10541–10544; [8] H. Kang, M. R. Alam, V. Dixit, M. Fisher, R. L. Juliano, Bioconjugate Chem. Angew. Chem. 2014, 126, 10711–10715. 12 2008, 19, 2182–2188. [32] J. Liu, A. Skradis, C. Kolar, J. Kolath, J. Anderson, T. Lawson, J. Talmadge, 13 W. H. Gmeiner, Nucleosides Nucleotides 1999, 18, 1789–1802. [9] X. Lu, F. Jia, X. Tan, D. Wang, X. Cao, J. Zheng, K. Zhang, J. Am. Chem. 14 Soc. 2016, 138, 9097–9100. [33] M. V. Céspedes, U. Unzueta, A. Aviñó, A. Gallardo, P. Álamo, R. Sala, A. [10] C. M. Niemeyer, T. Sano, C. L. Smith, C. R. Cantor, Nucleic Acids Res. 1994, Sánchez-Chardi, I. Casanova, M. A. Mangues, A. Lopez-Pousa, EMBO Mol. 15 22, 5530–5539. Med. 2018, 10, e8772. 16 [34] U. Unzueta, M. V. Cespedes, R. Sala, P. Alamo, A. Sánchez-Chardi, M. [11] R. P. Goodman, C. M. Erben, J. Malo, W. M. Ho, M. L. McKee, A. N. 17 Kapanidis, A. J. Turberfield, ChemBioChem, 2009, 10, 1551–1557. Pesarrodona, L. Sánchez-García, O. Cano-Garrido, A. Villaverde, E. [12] S. L. Khatwani, J. S. Kang, D. G. Mullen, M. A. Hast, L. S. Beese, M. D. Vázquez, J. Controlled Release 2018, 279, 29–39. 18 Distefano, T. A. Taton, Bioorg. Med. Chem. 2012, 20, 4532–4539. [35] R. Eritja, A. Pons, M. Escarceller, E. Giralt, F. Albericio, Tetrahedron 1991, 19 47, 4113–4120. [13] E. Jablonski, E. W. Moomaw, R. H. Tullis, J. L. Ruth, Nucleic Acids Res. 20 1986, 14, 6115–6128. [36] J. P. Leonetti, G. Degols, B. Lebleu, Bioconjugate Chem. 1990, 1, 149–153. [14] D. Corey, P. Schultz, Science 1987, 238, 1401–1403. [37] A. Levina, E. Mikhaleva, M. Repkova, V. Zarytova, Russ. J. Bioorg. Chem. 21 [15] J. Tominaga, Y. Kemori, Y. Tanaka, T. Maruyama, N. Kamiya, M. Goto, 2008, 34, 80–86. 22 [38] C.-H. Tung, K. J. Breslauer, S. Stein, Nucleic Acids Res. 1993, 21, 5489– Chem. Commun. 2007, 401–403. 23 [16] A. Dirksen, P. E. Dawson, Bioconjugate Chem. 2008, 19, 2543–2548. 5494. [17] M. Brinkley, Bioconjugate Chem. 1992, 3, 2–13. [39] J. Haralambidis, K. Angus, S. Pownall, L. Duncan, M. Chai, G. W. Tregear, 24 [18] H. Tsuchikama, Z. An, Protein Cell. 2018, 9, 33–46. Nucleic Acids Res. 1990, 18, 501–505. 25 [40] M. Lemaitre, B. Bayard, B. Lebleu, Proc. Natl. Acad. Sci. U. S. A., Early Ed. [19] F. Kukolka, C. M. Niemeyer, Org. Biomol. Chem. 2004, 2, 2203–2206. 26 [20] U. Unzueta, M. V. Céspedes, E. Vázquez, N. Ferrer-Miralles, R. Mangues, 1987, 84, 648–652. A. Villaverde, Trends Biotechnol. 2015, 33, 253–258. [41] M. Raouane, D. Desmaële, G. Urbinati, L. Massaad-Massade, P. Couvreur, 27 [21] F. Balkwill, Semin. Cancer Biol. Elsevier, 2004, 14, 171–179. Bioconjugate Chem. 2012, 23, 1091–1104. 28 [22] J. Kim, H. Takeuchi, S. T. Lam, R. R. Turner, H.-J. Wang, C. Kuo, L. Foshag, 29 A. J. Bilchik, D. S. Hoon, J. Clin. Oncol. 2005, 23, 2744–2753. 30 [23] Z. Liang, Y. Yoon, J. Votaw, M. M. Goodman, L. Williams, H. Shim, Cancer Res. 2005, 65, 967–971. Manuscript received: January 25, 2019 31 [24] X. Liang, Chem. Biol. Drug Des. 2008, 72, 97–110. Revised manuscript received: February 14, 2019 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 ChemistryOpen 2019, 8, 382 – 387 www.chemistryopen.org 387 © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA