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

Abstract

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.

Synthesis of Protein-Oligonucleotide Conjugates

“…The incorporation of non-native functional handles can either be achieved through bifunctional linker chemistry or through the incorporation of unnatural amino acids (UAAs). Either of these strategies can be used to incorporate azide reactive handles into protein sequences, including via the UAA pAzF (p-azidophenylalanine) [ 11 , 36 ]. This can then subsequently be exploited for copper-catalyzed azide-alkyne cycloaddition (CuAAC) [ 37 ] with an alkyne functionalized oligonucleotide or strain-promoted azide-alkyne cycloaddition (SPAAC) [ 11 , 24 , 38 , 39 ] by exploiting a strained cyclooctyne ( Figure 2 E).…”

“…Either of these strategies can be used to incorporate azide reactive handles into protein sequences, including via the UAA pAzF (p-azidophenylalanine) [ 11 , 36 ]. This can then subsequently be exploited for copper-catalyzed azide-alkyne cycloaddition (CuAAC) [ 37 ] with an alkyne functionalized oligonucleotide or strain-promoted azide-alkyne cycloaddition (SPAAC) [ 11 , 24 , 38 , 39 ] by exploiting a strained cyclooctyne ( Figure 2 E). Another commonly exploited UAA is p-acetylphenylalanine (pAcF) [ 7 , 31 ] which facilitates site specific incorporation of a ketone into a protein which can then undergo oxime ligation chemistry [ 32 , 40 ] with an alkoxyamine functionalized oligonucleotide ( Figure 2 F).…”

“…In addition to these readout applications protein-oligonucleotide conjugates can also be utilized for structural purposes. These predominantly involve the site-specific localization of target proteins to structures including DNA origami, [ 2 , 6 , 9 , 11 , 23 , 24 , 37 , 46 , 49 ] arrays, [ 35 ] nanoparticles [ 25 ] or even the surfaces of phages [ 41 ] to impart new activity. The oligonucleotide tags can also be used to bring labelled protein units together to form new entities with precise three-dimensional arrangements, [ 7 , 39 ] such as the characteristic Y-shape to be able to mimic antibody binding [ 26 , 40 ] or to recapitulate enzyme activity [ 22 , 28 , 33 , 45 ].…”

“…For these reasons nucleic acids are often used for specific recognition of partner strands enabling the formation of defined nano objects, or for the physical transfer of information through conformational change by acting as molecular circuitry. It is important to note that such systems can rely equally on native DNA or on designer nucleic acids (such as peptide nucleic acids (PNA) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ] or threoninol nucleic acids [ 11 ]) to fulfill this role. In contrast, proteins have a much wider array of potentially accessible structures as a result of their larger number of component building blocks, as well as the broader range of interactions these can undergo to influence global structure [ 1 ].…”

Supernatural: Artificial Nucleobases and Backbones to Program Hybridization-Based Assemblies and Circuits

“…27 This artificial backbone has also been used in assemblies generating triplexes, G-quadruplexes, 28 or imotifs, 29 as well as four-way junctions. 30 It was also used for unprecedented assemblies resulting in hexaplexes by leveraging artificial nucleobases designed to bind on two faces (Figure 1G). 31 Both XNAs (PNA and α-TNA) have been used to program the assembly of protein conjugates.…”