Targeted delivery of molecular spherical nucleic acids

During the last decades, oligonucleotides (ONs) have emerged as an alternative therapeutic modality for a variety of diseases. They can target disease mechanisms that are considered undruggable by small molecular drugs and, in comparison to small molecular drug development, they can be developed rapidly from target identification to clinical use. However, their therapeutic potential is hindered by rapid renal clearance, widespread biodistribution, and poor cellular uptake. Spherical nucleic acids (SNAs) are an alternative covalent formulation for the delivery of therapeutic ONs. They are large enough to avoid renal clearance and are readily taken up to various cell types. Despite many beneficial properties their targeted systemic delivery remains a challenge. Tissue specific ligands can be utilized to facilitate targeted delivery but commonly used methodologies for SNA synthesis provide poor control over the valency and site of ligand attachment. In this thesis, a controlled synthesis to obtain conjugates of well-defined [60]fullerene-based molecular SNAs (MSNAs) is described. The developed two-step assembly allows heterofunctionalization of MSNAs which can be utilized for site-specific ligand and labelling group integration. Various analytical methods were deployed to confirm the structural integrity and homogeneity of the synthesized MSNAs. Applicability of the MSNAs for site-specific radiolabelling was demonstrated and the effect of backbone chemistry, ligand decoration, and degree of labelling on the biodistribution was studied by positron emission tomography/computed tomography (PET/CT). The developed synthesis strategy was also applied for preparation of site-specific conjugates of glycan engineered antibodies (Ab) and MSNAs. Antigen binding properties, Ab-mediated endocytosis, and anti-proliferative effects of the conjugates on breast carcinoma cells were studied. Also, isopeptide bond formation between a peptide-ON conjugate and a recombinant protein was studied as an alternative methodology for generating Ab-ON conjugates (AOCs). The synthetic methodologies developed in this thesis can be used in the development of novel site-specific Ab-MSNA conjugates for diagnostic and therapeutic applications.Molekulaaristen pallonukleiinihappojen kohdennettu lääkeainekuljetus Viime vuosikymmeninä oligonukleotidit ovat nousseet esiin vaihtoehtoisena hoitomuotona useisiin sairauksiin. Niitä voidaan kohdistaa tautimekanismeihin, joihin pienmolekyylilääkkeet eivät sovellu, ja niiden kehitys kohteen tunnistamisesta kliiniseen käyttöön on nopeaa. Oligonukleotidien terapeuttista potentiaalia haittaavat kuitenkin nopea munuaissuodatus, laajalle levinnyt biodistribuutio ja huono soluunotto. Pallonukleiinihapot ovat terapeuttisten oligonukleotidien vaihtoehtoinen kovalenttinen formulaatio. Ne ovat riittävän suuria välttääkseen munuaissuodatuksen, ja ne otetaan helposti sisään erilaisiin solutyyppeihin. Monista hyödyllisistä ominaisuuksista huolimatta niiden haasteena on kohdennettu lääkeainekuljetus systeemisessä annostelussa. Kudosspesifisiä ligandeja voidaan käyttää parantamaan kohdennettua lääkeainekuljetusta, mutta yleisimmin käytetyt pallonukleiinihappojen synteesimenetelmät eivät mahdollista ligandien tai leima-aineiden paikkaspesifistä liittämistä. Tässä väitöskirjassa kuvataan [60]fullereenipohjaisten molekulaarisesti määriteltyjen pallonukleiinihappojen valmistusmenetelmä, joka mahdollistaa paikkaspesifisen ligandien ja leimausryhmien liittämisen osaksi rakennetta. Valmistettujen rakenteiden oikeellisuus ja yhteneväisyys varmistettiin useilla analyyttisillä menetelmillä. Pallonukleiinihapot radioleimattiin paikkaspesifisesti, ja oligonukleotidin selkärangan kemian, ligandikoristelun ja leimausasteen vaikutusta biodistribuutioon tutkittiin positroniemissiotomografialla/tietokonetomografialla (PET/CT). Työssä kuvataan myös glykaanimuokattujen vasta-aineiden ja pallonukeiinihappojen paikkaspesifisien konjugaattien valmistus. Lisäksi tutkittiin valmistettujen konjugaattien sitoutumisominaisuuksia kohdeantigeeniin, vasta-ainevälitteistä soluunottoa ja vaikutusta rintasyöpäsolujen kasvuun. Myös isopeptidisidoksen muodostumista peptidi-oligonukleotidikonjugaatin ja rekombinanttiproteiinin välillä tutkittiin vaihtoehtoisena menetelmänä vasta-aine-oligonukleotidikonjugaattien valmistamiseen. Kehitettyjä synteettisiä menetelmiä voidaan käyttää uudenlaisten paikkaspesifisten pallonukleiinihappojen ja vasta-aineiden yhdistelmärakenteiden kehittämisessä diagnostisiin ja terapeuttisiin sovelluksiin

19F-NMR-oligonukleotidikoettimet ja niiden sovellukset

19F NMR-spektroskopialla on osoittaunut tehokkaaksi työkaluksi erityisesti proteiininien ja nukleiinihappojen sekundäärirakenteiden transitioiden sekä niihin liittyvän dynamiikan ja pienligandi- ja makromolekyylivuorovaikutusten tutkimisessa. Erilaisia ja eri tartarkoituksiin sopivia leimaryhmiä tunnetaan kymmeniä, mikä mahdollistaa 19F NMR-spektroskopian käytön useissa DNA:n ja RNA:n sekundäärirakenteisiin liittyvissä sovelluksissa. Menetelmä perustuu oligonukleotideihin, joihin on kovalenttisesti liitetty fluorileima. NMR-spektroskopian avulla voidaan saada tietoa oligonukleotidien sekundäärirakenteiden paikallisista muutoksista. Tutkielman kirjallinen osa käsitettelee 19F NMR-oligonukleotikoettimissa käytettyjä erilaisia leimaryhmiä, niiden ominaisuuksia ja sovelluskohteita. Lisäksi käsitellään fluorattujen koettimien valmistamista ja verrataan NMR-spektroskopiaa muihin detektiomentelmiin, kuten UV-, CD- ja fluoresenssispektroskopiaan. 19F-NMR-spektroskopiaan perustuvilla koettimilla on sovelluspotentiaalia erityisesti diagnostisissa sovelluksissa. Useita olignukleotidien sekundäärirakenteisiin liittyviä ilmiöitä on pystytty tutkimaan in vivo, ja fluorileimaan perustuvien koettimien toimivuus on todennettu myös biologisesti merkittävissä pitoisuuksissa. Liittämällä hybridisaatioon muita kemiallisia ilmiöitä, voidaan fluorattujen koettimien herkkyyttä kasvattaa entisestään. Lisäksi lukuisat leimaryhmät mahdollistavat laajan kirjon erilaisia kohdesekvenssejä. Kokeellisessa osassa valmistettiin kaksi fluorattua C-nukleosidianalogia, joita on mahdollista käyttää leimaryhmänä 19F-NMR-oligonukleotidikoettimessa. Lisäksi tutkittiin koettimien soveltuvuutta elohopeavälitteisen emäspariutumisen tutkimiseen. Toisen nukleosidi analogin todettiin monomeeritasolla merkuroituvan. Tästä nukleosidista valmistettiin fosforamidiittirakenneyksikkö, jota voidaan käyttää 19F-NMR-oligonukleotidikoettimen valmistamiseen. Kyseisellä koettimella voidaan tutkia organometallivälitteisestä emäspariutumista 19F-NMR-spektroskopisesti. Asiasanat: 19F-NMR, RNA, DNA, PNA, nukleotidi, kahtaispysyvä, kolmoiskierre

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-beta-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 beta-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 beta-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T-0.5 ca 7 h) to hydrazone-based structures

2-Trifluoromethyl-6-mercurianiline Nucleotide, a Sensitive F-19 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 F-19 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 F-19 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 F-19 NMR spectroscopy

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 17β-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 17β-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.</p

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)-β-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 β-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. </p

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 C-60 core, with azide and tetrazine units. This orthogonally bifunctional C-60 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.Peer reviewe

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.</p

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 oligonucleo-tides. 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 <i>N</i>-(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-β-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 β-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 β-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