Synthesis of peptide-siRNA conjugates via internal sulfonylphosphoramidate modifications and evaluation of their in vitro activity.

Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide-siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide-siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3'-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.

Self-Assembly of Ultrasmall 3D Architectures of (l)-Acyclic Threoninol Nucleic Acids with High Thermal and Serum Stability.

The primary challenge of implementing DNA nanostructures in biomedical applications lies in their vulnerability to nuclease degradation and variations in ionic strength. Furthermore, the size minimization of DNA and RNA nanostructures is limited by the stability of the DNA and RNA duplexes. This study presents a solution to these problems through the use of acyclic (l)-threoninol nucleic acid (aTNA), an artificial acyclic nucleic acid, which offers enhanced resilience under physiological conditions. The high stability of homo aTNA duplexes enables the design of durable nanostructures with dimensions below 5 nm, previously unattainable due to the inherent instability of DNA structures. The assembly of a stable aTNA-based 3D cube and pyramid that involves an i-motif formation is demonstrated. In particular, the cube outperforms its DNA-based counterparts in terms of stability. We furthermore demonstrate the successful attachment of a nanobody to the aTNA cube using the favorable triplex formation of aTNA with ssDNA. The selective in vitro binding capability to human epidermal growth factor receptor 2 is demonstrated. The presented research presents the use of aTNA for the creation of smaller durable nanostructures for future medical applications. It also introduces a new method for attaching payloads to these structures, enhancing their utility in targeted therapies.

Reversible Protection and Targeted Delivery of DNA Origami with a Disulfide-Containing Cationic Polymer.

DNA nanostructures have considerable biomedical potential as intracellular delivery vehicles as they are highly homogeneous and can be functionalized with high spatial resolution. However, challenges like instability under physiological conditions, limited cellular uptake, and lysosomal degradation limit their use. This paper presents a bio-reducible, cationic polymer poly(cystaminebisacrylamide-1,6-diaminohexane) (PCD) as a reversible DNA origami protector. PCD displays a stronger DNA affinity than other cationic polymers. DNA nanostructures with PCD protection are shielded from low salt conditions and DNase I degradation and show a 40-fold increase in cell-association when linked to targeting antibodies. Confocal microscopy reveals a potential secondary cell uptake mechanism, directly delivering the nanostructures to the cytoplasm. Additionally, PCD can be removed by cleaving its backbone disulfides using the intracellular reductant, glutathione. Finally, the application of these constructs is demonstrated for targeted delivery of a cytotoxic agent to cancer cells, which efficiently decreases their viability. The PCD protective agent that is reported here is a simple and efficient method for the stabilization of DNA origami structures. With the ability to deprotect the DNA nanostructures upon entry of the intracellular space, the possibility for the use of DNA origami in pharmaceutical applications is enhanced.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Conjugation of chemical handles and functional moieties to DNA during solid phase synthesis with sulfonyl azides.

Labelling of oligonucleotides with dyes, targeting ligands, and other moieties has become ever more essential in life-sciences. Conventionally, modifications are introduced to oligonucleotides during solid phase synthesis by special phosphoramidites functionalised with a chemical handle or the desired functional group. In this work, we present a facile and inexpensive method to introduce modifications to oligonucleotides without the need for special phosphoramidites. Sulfonyl azides are applied to react with one or more selected phosphite intermediates during solid phase synthesis. We have prepared 11 sulfonyl azides with different chemical handles such as amine, azide, alkyne, and thiol, and we have further introduced functionalities such as pyrene, other dyes, photo-switchable azobenzenes, and a steroid. The method is compatible with current phosphoramidite-based automated oligonucleotide synthesis and serves as a simple alternative to the unstable and expensive special phosphoramidites currently used for conjugation to oligonucleotides.

Protein-Templated Reactions Using DNA-Antibody Conjugates.

DNA-templated chemical reactions have found wide applications in drug discovery, programmed multistep synthesis, nucleic acid detection, and targeted drug delivery. The control of these reactions has, however, been limited to nucleic acid hybridization as a means to direct the proximity between reactants. In this work a system capable of translating protein-protein binding events into a DNA-templated reaction which leads to the covalent formation of a product is introduced. Protein-templated reactions by employing two DNA-antibody conjugates that are both able to recognize the same target protein and to colocalize a pair of reactant DNA strands able to undergo a click reaction are achieved. Two individual systems, each responsive to human serum albumin (HSA) and human IgG, are engineered and it is demonstrated that, while no reaction occurs in the absence of proteins, both protein-templated reactions can occur simultaneously in the same solution without any inter-system crosstalk.

Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties.

Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.

Insertion of Chemical Handles into the Backbone of DNA during Solid-Phase Synthesis by Oxidative Coupling of Amines to Phosphites.

Conjugation of molecules or proteins to oligonucleotides can improve their functional and therapeutic capacity. However, such modifications are often limited to the 5' and 3' end of oligonucleotides. Herein, we report the development of an inexpensive and simple method that allows for the insertion of chemical handles into the backbone of oligonucleotides. This method is compatible with standardized automated solid-phase oligonucleotide synthesis, and relies on formation of phosphoramidates. A unique phosphoramidite is incorporated into a growing oligonucleotide, and oxidized to the desired phosphoramidate using iodine and an amine of choice. Azides, alkynes, amines, and alkanes have been linked to oligonucleotides via internally positioned phosphoramidates with oxidative coupling yields above 80 %. We show the design of phosphoramidates from secondary amines that specifically hydrolyze to the phosphate only at decreased pH. Finally, we show the synthesis of an antibody-DNA conjugate, where the oligonucleotide can be selectively released in a pH 5.5 buffer.

Automated Solid-Phase Oligo(disulfide) Synthesis.

A method for automated solid-phase synthesis of oligo(disulfide)s was developed. It is based on a synthetic cycle comprising removal of a protecting group from a resin-bound thiol followed by treatment with monomers containing a thiosulfonate as an activated precursor. For ease of purification and characterization, the disulfide oligomers were synthesized as extensions of oligonucleotides on an automated oligonucleotide synthesizer. Six different dithiol monomer building blocks were synthesized. Sequence-defined oligomers of up to seven disulfide units were synthesized and purified. The sequence of the oligomer was confirmed by tandem MS/MS analysis. One of the monomers contains a coumarin cargo that can be released by a thiol-mediated release mechanism. When the monomer was incorporated into an oligo(disulfide) and subjected to reducing conditions, the cargo was released under near-physiological conditions, which underlines the potential use of these molecules in drug delivery systems.

Chemical Conjugation to Less Targeted Proteinogenic Amino Acids.

Protein bioconjugates are in high demand for applications in biomedicine, diagnostics, chemical biology and bionanotechnology. Proteins are large and sensitive molecules containing multiple different functional groups and in particular nucleophilic groups. In bioconjugation reactions it can therefore be challenging to obtain a homogeneous product in high yield. Numerous strategies for protein conjugation have been developed, of which a vast majority target lysine, cysteine and to a lesser extend tyrosine. Likewise, several methods that involve recombinantly engineered protein tags have been reported. In recent years a number of methods have emerged for chemical bioconjugation to other amino acids and in this review, we present the progress in this area.

One-Step Conversion of NHS Esters to Reagents for Site-Directed Labeling of IgG Antibodies.

Antibody conjugates are extensively used for diagnostics and therapeutics, and as a tool for molecular biology. To prepare such conjugates N-hydroxysuccinimide (NHS) esters are most often used due to the straightforward experimental procedure and the commercial accessibility of the reagents. Such conjugates are however highly heterogeneous, since only the reactivity of the lysines determines the distribution of labels. This has inspired the development of methods that experimentally are as facile but produce conjugates of higher quality. Herein, we report the development of a reagent that can, in one step, be activated with an NHS ester of choice and subsequently can be directly used for site-directed labeling of antibodies. The reagent can be prepared in three synthetic steps and produces conjugates with similar ease as for NHS esters, however in a site-directed manner. We show that the reagent is quantitatively activated by a variety of NHS esters, and we use these to functionalize IgG1, IgG2, and IgG4 antibodies.

Preparation of Maleimide-Modified Oligonucleotides from the Corresponding Amines Using N-Methoxycarbonylmaleimide.

Oligonucleotide conjugates constitute a versatile tool for research and bioanalytical purposes. Often, such conjugates are prepared by reaction between a thiol on the protein with a maleimide-modified oligonucleotide. Unlike most other chemical handles the maleimide functionality cannot be introduced directly during the solid-phase oligonucleotide synthesis, and therefore the standard method to introduce the maleimide functionality is to react an amino-modified DNA with a heterobifunctional linker containing an activated ester and a maleimide. Here, we present an alternative method for preparation of maleimide and monobromomaleimide-modified oligonucleotides from the corresponding amine using N-methoxycarbonylmaleimide and N-methoxycarbonylbromomaleimide, respectively. In this method, no additional linker is attached to the oligonucleotide, as the maleimide functionality is formed directly on the existing amine. The maleimide can thereby be positioned close to the oligonucleotide, providing a high degree of control over the final construct. The reaction occurs in 30-60 min under alkaline conditions. Maleimide-modified oligonucleotides prepared in this manner were conjugated to bovine serum albumin, and the reaction shows comparable reactivity to the corresponding oligonucleotide modified using the 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) linker.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

Functionalized Acyclic (l)-Threoninol Nucleic Acid Four-Way Junction with High Stability In Vitro and In Vivo.

Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well-defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non-toxic, and non-immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l-threoninol nucleic acid (aTNA) to form a four-way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum-stable, shows no non-targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer-targeting and a serum half-life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.

Chemistries for DNA Nanotechnology.

The predictable nature of DNA interactions enables the programmable assembly of highly advanced 2D and 3D DNA structures of nanoscale dimensions. The access to ever larger and more complex structures has been achieved through decades of work on developing structural design principles. Concurrently, an increased focus has emerged on the applications of DNA nanostructures. In its nature, DNA is chemically inert and nanostructures based on unmodified DNA mostly lack function. However, functionality can be obtained through chemical modification of DNA nanostructures and the opportunities are endless. In this review, we discuss methodology for chemical functionalization of DNA nanostructures and provide examples of how this is being used to create functional nanodevices and make DNA nanostructures more applicable. We aim to encourage researchers to adopt chemical modifications as part of their work in DNA nanotechnology and inspire chemists to address current challenges and opportunities within the field.

A Wireframe DNA Cube: Antibody Conjugate for Targeted Delivery of Multiple Copies of Monomethyl Auristatin†E.

In recent years, several antibody drug conjugates (ADC) have been accepted by the FDA as therapeutics against cancer. It is well-known that control of drug-to-antibody ratio (DAR) is vital for the success of an ADC, which inspires the advancement of better and simpler methods for tight control of DAR. We present the development of an antibody DNA wireframe cube conjugate for precise control of DAR. The DNA wireframe cube consists of four single strands, which when folded present eight single stranded domains. One domain is bound to a monofunctionalized antibody DNA conjugate, and the seven others are attached to DNA functionalized with the potent tubulin inhibitor MMAE, thereby preparing an ADC with a DAR of precisely seven. The formation of the ADC is investigated by gel electrophoresis and atomic force microscopy. Lastly, the developed MMAE loaded ADC was used for targeted drug delivery in vitro.

A Reagent for Amine-Directed Conjugation to IgG1 Antibodies.

Functionalized antibodies are an indispensable resource for diagnosis, therapy and as a research tool for chemical biology. However, simpler and better methodologies are often required to improve the labeling of antibodies in terms of selectivity and scalability. Herein, we report the development of an easily available chemical reagent that allows site-directed labeling of native human IgG1 antibodies in good yield and mono-labeling selectivity. The salicylaldehyde moiety of the reagent reacts with surface exposed lysine residues to transiently form an iminium ion, and this positions a semi-reactive ester in proximity of a second lysine residue that reacts with the ester to form an amide. Interestingly, it appears that the formation of the iminium ion also has a significant activating effect of the ester. We use flow cytometry and bio-layer interferometry to confirm that the labeled antibodies retain antigen binding.

Two-Dimensional Coordination Networks from Cyclic Dipeptides.

Peptide-based biomimetic nanostructures and metal-organic coordination networks on surfaces are two promising classes of hybrid materials which have been explored recently. However, despite the great versatility and structural variability of natural and synthetic peptides, the two directions have so far not been merged in fabrication of metal-organic coordination networks using peptides as building blocks. Here we demonstrate that cyclic peptides can be used as ligands to form highly ordered, two-dimensional, peptide-based metal-organic coordination networks. The networks are formed on a Au(111) surface through coadsorption of cyclic dialanine with Cu-adatoms under Ultra-High Vacuum (UHV) conditions. Scanning Tunneling Microscopy (STM) in combination with X-ray Photoelectron spectroscopy (XPS) has been utilized to characterize the network structures at submolecular resolution and expound the chemical changes involved in network coordination. The networks involve a motif of three cyclic dialanine molecules coordinating to a central Cu-adatom. Interestingly the networks expose pores functionalized by the side chain of the cyclic peptide, suggesting a general method to form functionalized porous metal-organic networks on surfaces.

Introduction of an Aldehyde Handle on Nanobodies by Affinity-Guided Labeling.

Chemically modified antigen-binding proteins are widely applied for their targeting abilities in the fields of biotechnology, medicine, and diagnostics. However, the production of site-selectively modified proteins remains a challenge. Here, we have designed a chemical probe for the introduction of a reactive aldehyde on nanobodies by metal-complex-guided conjugation. The probe design allows for purification of the conjugates, and the aldehyde constitutes an efficient handle for further modification of the nanobodies. In vitro experiments confirmed the binding activity and selectivity of fluorescent conjugates toward the native antigen. Furthermore, the modification strategy allowed for production of a nanobody-drug conjugate that was active in vitro.

Disulphide-mediated site-directed modification of proteins.

Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.