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1.
ACS Cent Sci ; 9(5): 892-904, 2023 May 24.
Article in English | MEDLINE | ID: mdl-37252343

ABSTRACT

Nature has evolved intricate machinery to target and degrade RNA, and some of these molecular mechanisms can be adapted for therapeutic use. Small interfering RNAs and RNase H-inducing oligonucleotides have yielded therapeutic agents against diseases that cannot be tackled using protein-centered approaches. Because these therapeutic agents are nucleic acid-based, they have several inherent drawbacks which include poor cellular uptake and stability. Here we report a new approach to target and degrade RNA using small molecules, proximity-induced nucleic acid degrader (PINAD). We have utilized this strategy to design two families of RNA degraders which target two different RNA structures within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral pseudoknot. We demonstrate that these novel molecules degrade their targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy allows any RNA binding small molecule to be converted into a degrader, empowering RNA binders that are not potent enough to exert a phenotypic effect on their own. PINAD raises the possibility of targeting and destroying any disease-related RNA species, which can greatly expand the space of druggable targets and diseases.

2.
Bioconjug Chem ; 33(11): 2097-2102, 2022 11 16.
Article in English | MEDLINE | ID: mdl-36315583

ABSTRACT

Alkylating agents for nucleic acids have been widely used in cancer chemotherapy, as well as in chemical biology for strong inhibitors and tagging methods. We provide a series of reactive OFF-ON type alkylating agents which enable the reactivity modulation toward G-quadruplex (G4) DNA and RNA. Due to the protonation-accelerated process and equilibrium elimination method, the amine leaving groups show highly reactive and storable properties in an extensive investigation of vinyl quinazolinone (VQ) precursors with different leaving groups.


Subject(s)
Alkylating Agents , G-Quadruplexes , Alkylating Agents/chemistry , DNA/chemistry , RNA
3.
Org Biomol Chem ; 19(13): 2891-2894, 2021 04 07.
Article in English | MEDLINE | ID: mdl-33570069

ABSTRACT

The selective alkylation of nucleic acids is important for a medicinal approach and biological study. We now report a novel selective alkylation of the parallel G-quadruplex structure using the conjugate of the macrocyclic hexaoxazole L2G2-6OTD-1M1PA and vinyl-quinazolinone-S(O)Me (6OTD-VQ-S(O)Me).


Subject(s)
DNA/chemical synthesis , Macrocyclic Compounds/chemistry , Oxazoles/chemistry , Quinazolinones/chemistry , Vinyl Compounds/chemistry , Alkylation , DNA/chemistry , G-Quadruplexes , Molecular Structure
4.
ACS Cent Sci ; 6(12): 2196-2208, 2020 Dec 23.
Article in English | MEDLINE | ID: mdl-33376781

ABSTRACT

The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2'-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner. Here we describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, we have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide-alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we show that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation.

5.
Nucleic Acids Res ; 47(13): 6578-6589, 2019 07 26.
Article in English | MEDLINE | ID: mdl-31188442

ABSTRACT

Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these structures are involved in genetic control and diseases. Selective alkylation of these higher-order structures is challenging due to the chemical instability of the alkylating agent and side-reactions with the single- or double-strand DNA and RNA. We now report the reactive OFF-ON type alkylating agents, vinyl-quinazolinone (VQ) precursors with a sulfoxide, thiophenyl or thiomethyl group for the OFF-ON control of the vinyl reactivity. The stable VQ precursors conjugated with aminoacridine, which bind to the G-4 DNA, selectively reacted with a T base on the G-4 DNA in contrast to the single- and double-strand DNA. Additionally, the VQ precursor reacted with the T or U base in the AP-site, G-4 RNA and T-T mismatch structures. These VQ precursors would be a new candidate for the T or U specific alkylation in the higher-ordered structures of nucleic acids.


Subject(s)
Alkylating Agents/pharmacology , DNA/drug effects , Nucleic Acid Conformation/drug effects , Alkylating Agents/chemical synthesis , Alkylating Agents/chemistry , Alkylation , Base Pairing , DNA/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/drug effects , G-Quadruplexes/drug effects , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Purines/chemistry , Purines/pharmacology , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Triazines/chemistry , Triazines/pharmacology , Vinyl Compounds/chemistry , Vinyl Compounds/pharmacology
6.
Bioorg Med Chem ; 26(12): 3551-3558, 2018 07 23.
Article in English | MEDLINE | ID: mdl-29807700

ABSTRACT

Higher-order structures of nucleic acids have become widely noted for their biological consequences and the discovery of an alkylating small molecule for these structures has been of interest due to its therapeutic potential. We previously developed the vinyldiaminotriazine (VDAT)-acridine conjugate as a T-T mismatch alkylating agent. In this report, we focused on the finding of the alkylation to the G-quadruplex (G4) DNA with VDAT-acridine conjugates. The VDAT-acridine conjugates exhibited a considerable alkylation ability to G4 under mild conditions. Moreover, the investigation of properties with the alkylated G4 revealed that alkylation by this conjugate significantly increased the stability of the G4 structure. This study provides a starting point in the further development of selective G4 alkylating small molecules.


Subject(s)
Acridines/chemistry , Alkylating Agents/chemistry , G-Quadruplexes , Triazines/chemistry , Alkylation , Base Pair Mismatch , Base Sequence , Thymine/chemistry , Thymine/metabolism , Transition Temperature
7.
Org Biomol Chem ; 16(9): 1436-1441, 2018 02 28.
Article in English | MEDLINE | ID: mdl-29412214

ABSTRACT

The G-quadruplex structure has been found in biologically significant regions of the genomic DNA, including the telomere and promoter regions, and is known to play an important role in a number of biological processes. In this paper, we report the development of alkylating probes for the G-quadruplex structure and evaluation of the properties of the modified G-quadruplex structure.


Subject(s)
DNA/chemistry , G-Quadruplexes , Alkylation , Base Sequence , DNA/genetics , Telomere/genetics
8.
Nucleic Acids Res ; 46(3): 1059-1068, 2018 02 16.
Article in English | MEDLINE | ID: mdl-29309639

ABSTRACT

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.


Subject(s)
Acridines/chemistry , Base Pair Mismatch , DNA Replication , DNA/chemistry , Thymidine/chemistry , Triazines/chemistry , Vinyl Compounds/chemistry , Alkylation , Base Pairing , Base Sequence , DNA/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Humans , Molecular Targeted Therapy , Myotonic Dystrophy/genetics , Myotonic Dystrophy/pathology , Myotonic Dystrophy/therapy , Thymidine/metabolism , Transcription, Genetic , Trinucleotide Repeats
9.
Bioorg Med Chem ; 25(7): 2191-2199, 2017 04 01.
Article in English | MEDLINE | ID: mdl-28268052

ABSTRACT

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.


Subject(s)
Nucleic Acid Conformation , RNA, Double-Stranded/chemical synthesis , Chromatography, High Pressure Liquid , Circular Dichroism , RNA, Double-Stranded/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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