11 research outputs found
Synthesis of RNA 5′-Azides from 2′‑<i>O</i>‑Pivaloyloxymethyl-Protected RNAs and Their Reactivity in Azide–Alkyne Cycloaddition Reactions
Commercially available
2′-<i>O</i>-pivaloyloxymethyl
(PivOM) phosphoramidites were employed in an SPS protocol for RNA
5′ azides. The utility of the N<sub>3</sub>-RNAs in CuAAC and
SPAAC was demonstrated by RNA 5′ labeling, chemical ligation
including fragment joining and cyclization, and bioconjugation. As
a result, several new RNA conjugates that may be valuable tools for
studies on biological events such as innate immune response (cyclic
dinucleotides), post-transcriptional gene regulation (circular RNAs),
or mRNA turnover (m<sup>7</sup>G capped RNAs) were obtained
Preparation of Synthetically Challenging Nucleotides Using Cyanoethyl P‑Imidazolides and Microwaves
We describe a general method for the elongation of nucleoside oligophosphate chains by means of cyanoethyl (CE) phosphorimidazolides. Though the method requires a phosphorylation and subsequent deprotection reaction, both steps could be achieved in one pot without isolation/purification of the initial phosphorylation product. We have also found that pyrophosphate bond formation by this method is significantly accelerated by microwave irradiation
Ethynyl, 2‑Propynyl, and 3‑Butynyl C‑Phosphonate Analogues of Nucleoside Di- and Triphosphates: Synthesis and Reactivity in CuAAC
The
synthesis and reactivity of a novel class of clickable nucleotide
analogues containing a C-phosphonate subunit that has an alkyne group
at the terminal position of the oligophosphate chain are reported.
The C-phosphonate subunits were prepared by simple one- or two-step
procedures using commercially available reagents. Nucleotides were
prepared by MgCl<sub>2</sub>-catalyzed coupling reactions and then
subjected to CuAAC reactions with various azide compounds to afford
5′-γ-labeled nucleoside triphosphates in excellent yields
Synthesis of Fluorophosphate Nucleotide Analogues and Their Characterization as Tools for <sup>19</sup>F NMR Studies
To broaden the scope of existing
methods based on <sup>19</sup>F nucleotide labeling, we developed
a new method for the synthesis
of fluorophosphate (oligo)nucleotide analogues containing an O to
F substitution at the terminal position of the (oligo)phosphate moiety
and evaluated them as tools for <sup>19</sup>F NMR studies. Using
three efficient and comprehensive synthetic approaches based on phosphorimidazolide
chemistry and tetra-<i>n</i>-butylammonium fluoride, fluoromonophosphate,
or fluorophosphate imidazolide as fluorine sources, we prepared over
30 fluorophosphate-containing nucleotides, varying in nucleobase type
(A, G, C, U, m<sup>7</sup>G), phosphate chain length (from mono to
tetra), and presence of additional phosphate modifications (thio,
borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating
reagent for 5′-phosphorylated oligos we also synthesized oligonucleotide
5′-(2-fluorodiphosphates), which are potentially useful as <sup>19</sup>F NMR hybridization probes. The compounds were characterized
by <sup>19</sup>F NMR and evaluated as <sup>19</sup>F NMR molecular
probes. We found that fluorophosphate nucleotide analogues can be
used to monitor activity of enzymes with various specificities and
metal ion requirements, including human DcpS enzyme, a therapeutic
target for spinal muscular atrophy. The compounds can also serve as
reporter ligands for protein binding studies, as exemplified by studying
interaction of fluorophosphate mRNA cap analogues with eukaryotic
translation initiation factor (eIF4E)
Magnetic-Nanoparticle-Decorated Polypyrrole Microvessels: Toward Encapsulation of mRNA Cap Analogues
Many phosphorylated nucleoside derivatives
have therapeutic potential,
but their application is limited by problems with membrane permeability
and with intracellular delivery. Here, we prepared polypyrrole microvessel
structures modified with superparamagnetic nanoparticles for use as
potential carriers of nucleotides. The microvessels were prepared
via the photochemical polymerization of the monomer onto the surface
of aqueous ferrofluidic droplets. A complementary physicochemical
analysis revealed that a fraction of the nanoparticles was embedded
in the microvessel walls, while the other nanoparticles were in the
core of the vessel. SQUID (superconducting quantum interference device)
measurements indicated that the incorporated nanoparticles retained
their superparamagnetic properties; thus, the resulting nanoparticle-modified
microvessels can be directed by an external magnetic field. As a result
of these features, these microvessels may be useful as drug carriers
in biomedical applications. To demonstrate the encapsulation of drug
molecules, two labeled mRNA cap analogues, nucleotide-derived potential
anticancer agents, were used. It was shown that the cap analogues
are located in the aqueous core of the microvessels and can be released
to the external solution by spontaneous permeation through the polymer
walls. Mass spectrometry analysis confirmed that the cap analogues
were preserved during encapsulation, storage, and release. This finding
provides a foundation for the future development of anticancer therapies
and for the delivery of nucleotide-based therapeutics
Magnetic-Nanoparticle-Decorated Polypyrrole Microvessels: Toward Encapsulation of mRNA Cap Analogues
Many phosphorylated nucleoside derivatives
have therapeutic potential,
but their application is limited by problems with membrane permeability
and with intracellular delivery. Here, we prepared polypyrrole microvessel
structures modified with superparamagnetic nanoparticles for use as
potential carriers of nucleotides. The microvessels were prepared
via the photochemical polymerization of the monomer onto the surface
of aqueous ferrofluidic droplets. A complementary physicochemical
analysis revealed that a fraction of the nanoparticles was embedded
in the microvessel walls, while the other nanoparticles were in the
core of the vessel. SQUID (superconducting quantum interference device)
measurements indicated that the incorporated nanoparticles retained
their superparamagnetic properties; thus, the resulting nanoparticle-modified
microvessels can be directed by an external magnetic field. As a result
of these features, these microvessels may be useful as drug carriers
in biomedical applications. To demonstrate the encapsulation of drug
molecules, two labeled mRNA cap analogues, nucleotide-derived potential
anticancer agents, were used. It was shown that the cap analogues
are located in the aqueous core of the microvessels and can be released
to the external solution by spontaneous permeation through the polymer
walls. Mass spectrometry analysis confirmed that the cap analogues
were preserved during encapsulation, storage, and release. This finding
provides a foundation for the future development of anticancer therapies
and for the delivery of nucleotide-based therapeutics
Magnetic-Nanoparticle-Decorated Polypyrrole Microvessels: Toward Encapsulation of mRNA Cap Analogues
Many phosphorylated nucleoside derivatives
have therapeutic potential,
but their application is limited by problems with membrane permeability
and with intracellular delivery. Here, we prepared polypyrrole microvessel
structures modified with superparamagnetic nanoparticles for use as
potential carriers of nucleotides. The microvessels were prepared
via the photochemical polymerization of the monomer onto the surface
of aqueous ferrofluidic droplets. A complementary physicochemical
analysis revealed that a fraction of the nanoparticles was embedded
in the microvessel walls, while the other nanoparticles were in the
core of the vessel. SQUID (superconducting quantum interference device)
measurements indicated that the incorporated nanoparticles retained
their superparamagnetic properties; thus, the resulting nanoparticle-modified
microvessels can be directed by an external magnetic field. As a result
of these features, these microvessels may be useful as drug carriers
in biomedical applications. To demonstrate the encapsulation of drug
molecules, two labeled mRNA cap analogues, nucleotide-derived potential
anticancer agents, were used. It was shown that the cap analogues
are located in the aqueous core of the microvessels and can be released
to the external solution by spontaneous permeation through the polymer
walls. Mass spectrometry analysis confirmed that the cap analogues
were preserved during encapsulation, storage, and release. This finding
provides a foundation for the future development of anticancer therapies
and for the delivery of nucleotide-based therapeutics
Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins
Gα
proteins as part of heterotrimeric G proteins are molecular
switches essential for G protein-coupled receptor- mediated intracellular
signaling. The role of the Gα subunits has been examined for
decades with various guanine nucleotides to elucidate the activation
mechanism and Gα protein-dependent signal transduction. Several
approaches describe fluorescent ligands mimicking the GTP function,
yet lack the efficient estimation of the proteins’ GTP binding
activity and the fraction of active protein. Herein, we report the
development of a reliable fluorescence anisotropy-based method to
determine the affinity of ligands at the GTP-binding site and to quantify
the fraction of active Gαi1 protein. An advanced bacterial expression
protocol was applied to produce active human Gαi1 protein, whose
GTP binding capability was determined with novel fluorescently labeled
guanine nucleotides acting as high-affinity Gαi1 binders compared
to the commonly used BODIPY FL GTPγS. This study thus contributes
a new method for future investigations of the characterization of
Gαi and other Gα protein subunits, exploring their corresponding
signal transduction systems and potential for biomedical applications
Trinucleotide mRNA Cap Analogue <i>N</i>6‑Benzylated at the Site of Posttranscriptional <sup>m6</sup>A<sub>m</sub> Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo
Eukaryotic mRNAs undergo cotranscriptional 5′-end
modification
with a 7-methylguanosine cap. In higher eukaryotes, the cap carries
additional methylations, such as m6Ama
common epitranscriptomic mark unique to the mRNA 5′-end. This
modification is regulated by the Pcif1 methyltransferase and the FTO
demethylase, but its biological function is still unknown. Here, we
designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap–m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped
transcripts. The Bn6Am moiety was shown to act
as a reversed-phase high-performance liquid chromatography (RP-HPLC)
purification handle, allowing the separation of capped and uncapped
RNA species, and to produce transcripts with lower dsRNA content than
reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent.
m7GpppBn6AmpG-capped mRNAs encoding
reporter proteins administered intravenously to mice provided up to
6-fold higher protein outputs than reference mRNAs, while mRNAs encoding
tumor antigens showed superior activity in therapeutic settings as
anticancer vaccines. The biochemical characterization suggests several
phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions,
(ii) involvement in alternative translation initiation, and (iii)
subtle differences in mRNA impurity profiles or a combination of these
effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines
and therapeutics and serve as molecular tools to unravel the role
of m6Am in mRNA
Trinucleotide mRNA Cap Analogue <i>N</i>6‑Benzylated at the Site of Posttranscriptional <sup>m6</sup>A<sub>m</sub> Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo
Eukaryotic mRNAs undergo cotranscriptional 5′-end
modification
with a 7-methylguanosine cap. In higher eukaryotes, the cap carries
additional methylations, such as m6Ama
common epitranscriptomic mark unique to the mRNA 5′-end. This
modification is regulated by the Pcif1 methyltransferase and the FTO
demethylase, but its biological function is still unknown. Here, we
designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap–m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped
transcripts. The Bn6Am moiety was shown to act
as a reversed-phase high-performance liquid chromatography (RP-HPLC)
purification handle, allowing the separation of capped and uncapped
RNA species, and to produce transcripts with lower dsRNA content than
reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent.
m7GpppBn6AmpG-capped mRNAs encoding
reporter proteins administered intravenously to mice provided up to
6-fold higher protein outputs than reference mRNAs, while mRNAs encoding
tumor antigens showed superior activity in therapeutic settings as
anticancer vaccines. The biochemical characterization suggests several
phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions,
(ii) involvement in alternative translation initiation, and (iii)
subtle differences in mRNA impurity profiles or a combination of these
effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines
and therapeutics and serve as molecular tools to unravel the role
of m6Am in mRNA