In Vivo Imaging of [60]Fullerene-Based Molecular Spherical Nucleic Acids by Positron Emission Tomography.

18F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factor receptor 2 (HER2) mRNA antisense oligonucleotide sequence with a native phosphodiester and phosphorothioate backbone, were synthesized, site-specifically labeled with a positron emitting fluorine-18 and intravenously administrated via tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistribution of the MSNAs was monitored in vivo by positron emission tomography/computed tomography (PET/CT) imaging. MSNA with a native phosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediated degradation, whereas the corresponding phosphorothioate analogue (MSNA-PS) with improved enzymatic stability showed an interesting biodistribution profile in vivo. One hour after the injection, majority of the radioactivity was observed in spleen and liver but also in blood with an average tumor-to-muscle ratio of 2. The prolonged radioactivity in blood circulation may open possibilities to the targeted delivery of the MSNAs.

2-Trifluoromethyl-6-mercurianiline Nucleotide, a Sensitive 19F NMR Probe for Hg(II)-mediated Base Pairing.

A 2-trifluoromethylaniline C-nucleoside was synthesized, incorporated in the middle of an oligonucleotide, and mercurated. The affinity of the mercurated oligonucleotide toward complementary strands placing each of the canonical nucleobases opposite to the organomercury nucleobase analogue was examined by ultraviolet (UV), circular dichroism (CD), and 19F NMR spectroscopy analyses. According to the UV melting profile analysis, the organomercury nucleobase analogue showed increased affinities in the order T > G > C > A. The CD profiles indicated the typical B-type helix in each case. The 19F resonance signal proved sensitive for the local environmental changes, showing clearly distinct signals for the duplexes with different opposing nucleobases. Furthermore, valuable information on the mercurated oligonucleotide and its binding to complementary strands at varying temperature could be obtained by 19F NMR spectroscopy.

Controlled Monofunctionalization of Molecular Spherical Nucleic Acids on a Buckminster Fullerene Core.

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs.

Expanding the Scope of the Cleavable N-(methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates.

Oligonucleotides modified by a 2'-deoxy-2'-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-ß-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a ß-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to ß-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T0.5 ca 7 h) to hydrazone-based structures.

Synthesis of Site-Specific Antibody-[60]Fullerene-Oligonucleotide Conjugates for Cellular Targeting.

An ideal therapeutic antibody-oligonucleotide conjugate (AOC) would be a uniform construct, contain a maximal oligonucleotide (ON) payload, and retain the antibody (Ab)-mediated binding properties, which leads to an efficient delivery of the ON cargo to the site of therapeutic action. Herein, [60]fullerene-based molecular spherical nucleic acids (MSNAs) have been site-specifically conjugated to antibodies (Abs), and the Ab-mediated cellular targeting of the MSNA-Ab conjugates has been studied. A well-established glycan engineering technology and robust orthogonal click chemistries yielded the desired uniform MSNA-Ab conjugates (MW ∼ 270 kDa), with an oligonucleotide (ON):Ab ratio of 24:1, in 20-26% isolated yields. These AOCs retained the antigen binding properties (Trastuzumab's binding to human epidermal growth factor receptor 2, HER2), studied by biolayer interferometry. In addition, Ab-mediated endocytosis was demonstrated with live-cell fluorescence and phase-contrast microscopy on BT-474 breast carcinoma cells, overexpressing HER2. The effect on cell proliferation was analyzed by label-free live-cell time-lapse imaging.

Tetrazine Glycoconjugate for Pretargeted Positron Emission Tomography Imaging of trans-Cyclooctene-Functionalized Molecular Spherical Nucleic Acids.

Pretargeted concept in positron emission tomography (PET) together with bioorthogonal chemistry is an elegant solution to study processes with slow pharmacokinetics by utilizing radiotracers labeled with short-lived radionuclides. Namely, radiotracers based on tetrazine ligation with trans-cyclooctene (TCO) via the inverse electron demand Diels-Alder (IEDDA) reaction have become a state-of-the-art for the pretargeted PET imaging. For radiolabeling of tetrazine scaffolds, indirect radiofluorination methods are often preferred, as tetrazines are vulnerable to harsh conditions typically necessary for the direct radiofluorination. 18F-Fluoroglycosylation is an indirect radiofluorination method, which allows the introduction of a widely accessible glucose analog 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) to aminooxy-functionalized precursors via oxime formation. Here, we report the biological evaluation of [18F]FDG-Tz as a tracer for pretargeted PET imaging of TCO-functionalized molecular spherical nucleic acids (MSNA) against human epidermal growth factor receptor 2 (HER2) mRNA. The oxime ether formation between [18F]FDG and tetrazine oxyamine resulted in [18F]FDG-Tz with high radiochemical purity (>99%) and moderate yields (6.5 ± 3.6%, n = 5). Biological evaluation of [18F]FDG-Tz in healthy mice indicated favorable pharmacokinetics with quick blood clearance, urinary excretion as the main elimination route, and the absence of GLUT1 transportation. The successful pretargeted experiments with TCO-functionalized MSNA revealed higher tumor uptake compared to preclicked MSNA in HER2-expressing human breast cancer xenograft-bearing mice.

Synthesis of an Azide- and Tetrazine-Functionalized [60]Fullerene and Its Controlled Decoration with Biomolecules.

Bingel cyclopropanation between Buckminster fullerene and a heteroarmed malonate was utilized to produce a hexakis-functionalized C60 core, with azide and tetrazine units. This orthogonally bifunctional C60 scaffold can be selectively one-pot functionalized by two pericyclic click reactions, that is, inverse electron-demand Diels-Alder and azide-alkyne cycloaddition, which with appropriate ligands (monosaccharides, a peptide and oligonucleotides tested) allows one to control the assembly of heteroantennary bioconjugates.

Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR.

Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules for therapeutics and biotechnological applications. Most widely employed chemical labeling methods for proteins are based on targeting of Lys or Cys residues that leads to mixed stoichiometry in the degree of conjugation and may interfere with antigen binding, thus, compromising the function of the antibody. A site-specific oligonucleotide conjugation technology providing full control over valency in mild reaction conditions would be an advancement to the state-of-the-art in bioconjugation. Herein, we demonstrate the production of single-chain variable fragment antibodies with fused SpyCatcher (scFv-SpyCatcher, monovalent) and alkaline phosphatase-SpyCatcher (scFv-AP-SpyCatcher, bivalent) on C-terminus and their conjugation to SpyTag002-oligonucleotide in phosphate-buffered saline (PBS). The formation of a covalent isopeptide bond between the protein and SpyTag002-oligonucleotide was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the functionality of the obtained AOCs was confirmed in immuno-polymerase chain reaction (PCR) assays for the detection of microcystin-LR and 17ß-estradiol. Based on time-resolved fluorescence immunoassays with scFv-AP fusion constructs, we observed that the SpyCatcher and SpyCatcher-SpyTag002-oligonucleotide part lowered the absolute signal obtained from the assay by 27.6 and 48.4% at 2 nM and by 26.2 and 27.6% at 100 pM microcystin-LR and 17ß-estradiol concentrations, respectively. Nevertheless, the overall sensitivity of the immuno-PCR assays was similar to the time-resolved fluorescence immunoassays performed with the same components. In this study, vectors for SpyCatcher-fusion construction were created for directional cloning with SfiI sites enabling the rapid generation of AOC constructs for site-specific SpyTag-oligonucleotide conjugation.

Immobilized Carbohydrates for Preparation of 3'-Glycoconjugated Oligonucleotides.

A detailed protocol for preparation 3'-glycoconjugated oligonucleotides is described based on one-pot immobilization of 4,4'-dimethoxytrityl-protected carbohydrates to a solid support followed by on-support peracetylation and automated oligonucleotide assembly. Compared to an appropriate building block approach and post-synthetic manipulation of oligonucleotides, this protocol may simplify the synthesis scheme and increase overall yield of the conjugates. Furthermore, the immobilization to a solid support typically increases the stability of reactants, enabling prolonged storage, and makes subsequent processing convenient. Automated assembly on these carbohydrate-modified supports using conventional phosphoramidite chemistry produces 3'-glycoconjugated oligonucleotides in relatively high yield and purity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 1-O-tert-butyldimethylsilyl-6-O-(4,4'-dimethoxytrityl)-ß-D-glucose Basic Protocol 2: Synthesis of 6-O-dimethoxytrityl-2,3,1',3',4',6'-hexa-O-benzoylsucrose Basic Protocol 3: Synthesis of 6″-O-dimethoxytrityl-N-trifluoroacetyl-protected aminoglycosides Basic Protocol 4: Synthesis of 3-O-dimethoxytrityl-propyl ß-D-galactopyranoside Basic Protocol 5: Synthesis of trivalent N-acetyl galactosamine cluster Basic Protocol 6: Synthesis of carbohydrate monosuccinates and their immobilization to a solid support Basic Protocol 7: Oligonucleotide synthesis using immobilized carbohydrates.