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1.
Chembiochem ; : e202400417, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38923227

RESUMO

MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti-miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2-amino-6-vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross-linked with AVP-conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross-linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7-propynyl group - which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP - a deoxy analog of ADpVP - through a simplified synthesis protocol. Evaluation of the cross-linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross-linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross-linking nucleosides for indirect miRNA function inhibition.

2.
Bioorg Med Chem Lett ; 109: 129855, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38908766

RESUMO

The role of G-quadruplex (G4) in cellular processes can be investigated by the covalent modification of G4-DNA using alkylating reagents. Controllable alkylating reagents activated by external stimuli can react elegantly and selectively. Herein, we report a chemical activation system that can significantly boost the reaction rate of methylamine-protected vinyl-quinazolinone (VQ) derivative for the alkylation of G4-DNA. The two screened activators can transform low-reactive VQ-NHR' to highly reactive intermediates following the Michael addition mechanism. This approach expands the toolbox of activable G4 alkylating reagents.


Assuntos
Quadruplex G , Metilaminas , Quinazolinonas , Alquilação , Quadruplex G/efeitos dos fármacos , Metilaminas/química , Metilaminas/farmacologia , Metilaminas/síntese química , Quinazolinonas/química , Quinazolinonas/farmacologia , Quinazolinonas/síntese química , Humanos , Estrutura Molecular , DNA/química , Compostos de Vinila/química , Compostos de Vinila/farmacologia
3.
Bioconjug Chem ; 2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36930464

RESUMO

The formation of interlocked structures, such as rotaxane and catenane, enables noncovalent conjugations. We previously confirmed that the chemically cyclized pseudorotaxane-forming oligodeoxynucleotides (prfODNs) with double-tailed parts formed a pseudorotaxane structure with the target DNA and RNA via the slipping process. Here, we report the one-step synthesis of cyclized prfODNs from alkyne-modified ODNs, after which we investigated the properties and mechanism of the slipping process and performed noncovalent RNA labeling with prfODNs. Additionally, the catenane structure was formed by the combination of pseudorotaxane formation with a 5'-end-phosphorylated RNA and enzymatic ligation. The newly synthesized prfODN represents a new tool for achieving the noncovalent conjugation of various functional moieties to RNAs.

4.
Chem Rec ; 23(2): e202200194, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36111635

RESUMO

DNA and RNA can adopt a variety of stable higher-order structural motifs, including G-quadruplex (G4 s), mismatches, and bulges. Many of these secondary structures are closely related to the regulation of gene expression. Therefore, the higher-order structure of nucleic acids is one of the candidate therapeutic targets, and the development of binding molecules targeting the higher-order structure of nucleic acids has been pursued vigorously. Furthermore, as one of the methodologies for detecting the higher-order structures of these nucleic acids, developing techniques for the selective chemical modification of the higher-order structures of nucleic acids is also underway. In this personal account, we focus on the following higher-order structures of nucleic acids, double-stranded DNA containing the abasic site, T-T/U-U mismatch structure, and G-quadruplex structure, and describe the development of molecules that bind to and chemically modify these structures.


Assuntos
Quadruplex G , Ácidos Nucleicos , DNA/química , RNA/química
5.
Bioconjug Chem ; 33(11): 2097-2102, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36315583

RESUMO

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.


Assuntos
Alquilantes , Quadruplex G , Alquilantes/química , DNA/química , RNA
6.
J Org Chem ; 87(5): 2267-2276, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-34978198

RESUMO

Specific chemical reactions by enzymes acting on a nucleobase are realized by flipping the target base out of the helix. Similarly, artificial oligodeoxynucleotides (ODNs) can also induce the base flipping and a specific chemical reaction. We now report an easily prepared and unique structure-providing photo-cross-linking reaction by taking advantage of the base-flipping-out field formed by alkene-type base-flipping-inducing artificial bases. Two 3-arylethenyl-5-methyl-2-pyridone nucleosides with the Ph or An group were synthesized and incorporated into the ODNs. We found that the two Ph derivatives provided the cross-linked product in a high yield only by a 10 s photoirradiation when their alkenes overlap each other in the duplex DNA. The highly efficient reaction enabled forming a cross-linked product even when using the duplex with a low Tm value.


Assuntos
Alcenos , DNA , Conformação de Ácido Nucleico , Nucleosídeos , Oligodesoxirribonucleotídeos
7.
Org Biomol Chem ; 20(23): 4699-4708, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35622064

RESUMO

The interstrand crosslinking and threaded structures of nucleic acids have high potential in oligonucleotide therapeutics, chemical biology, and nanotechnology. For example, properly designed crosslinking structures provide high activity and nuclease resistance for anti-miRNAs. The noncovalent labeling and modification by the threaded structures are useful as new chemical biology tools. Photoreversible crosslinking creates smart materials, such as reversible photoresponsive gels and DNA origami objects. This review introduces the creation of interstrand crosslinking and threaded structures, such as catenanes and rotaxanes, based on hybridization-specific chemical reactions and their functions and perspectives.


Assuntos
Ácidos Nucleicos , Rotaxanos , DNA/química , Nanotecnologia , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Rotaxanos/química
8.
Bioorg Med Chem Lett ; 48: 128257, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34246752

RESUMO

The interstrand crosslinking of nucleic acids is one of the strategies to create the stable complex between an oligonucleotide and RNA by covalent bond formation. We previously reported that fully 2'-O-methylated (2'-OMe) RNAs having the 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. In this study, we established a chemical method to efficiently synthesize the crosslinked 2'-OMe RNA duplexes using AVP and prepared the anti-miRNA oligonucleotides (AMOs) containing the antisense targeting miR-21 and crosslinked duplex at the terminal sequences. These AMOs showed a markedly higher anti miRNA activity than that of the commercially-available miR-21 inhibitor which has locked nucleic acid (LNA) residues.


Assuntos
MicroRNAs/antagonistas & inibidores , RNA/farmacologia , Relação Dose-Resposta a Droga , Humanos , Metilação , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Purinas/química , Purinas/farmacologia , RNA/síntese química , RNA/química , Relação Estrutura-Atividade , Compostos de Vinila/química , Compostos de Vinila/farmacologia
9.
Org Biomol Chem ; 19(13): 2891-2894, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33570069

RESUMO

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).


Assuntos
DNA/síntese química , Compostos Macrocíclicos/química , Oxazóis/química , Quinazolinonas/química , Compostos de Vinila/química , Alquilação , DNA/química , Quadruplex G , Estrutura Molecular
10.
Nucleic Acids Res ; 47(13): 6578-6589, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31188442

RESUMO

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.


Assuntos
Alquilantes/farmacologia , DNA/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Alquilantes/síntese química , Alquilantes/química , Alquilação , Pareamento de Bases , DNA/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Purinas/química , Purinas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Triazinas/química , Triazinas/farmacologia , Compostos de Vinila/química , Compostos de Vinila/farmacologia
11.
Nucleic Acids Res ; 46(17): 8710-8719, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30260454

RESUMO

Interlocked structures, such as rotaxane and catenane, combine both static and dynamic properties. To expand their unique properties into the chemical biology field, a spontaneous formation method of the interlocked structures with the target would be ideal. We have previously developed a pseudorotaxane-forming oligo DNA (prfODN) to spontaneously form topological DNA/RNA architectures. In this study, we report the structural optimization of prfODNs for the efficient and stable complex formation. The optimized prfODNs efficiently formed pseudorotaxane structures with a DNA or RNA target, and the yield for the RNA target reached 85% in 5 min. In addition, the optimized prfODNs could form the pseudorotaxane structure with a smaller ring size and the structure significantly increased the kinetic stability. Furthermore, the catenane structure was successfully formed with the optimized prfODNs to provide the conclusive evidence for the formation of the threaded structure. This information will be valuable for developing new chemical methods using functional nucleic acids for antisense oligo nucleotides and DNA/RNA nanotechnology.


Assuntos
Antracenos/química , DNA/química , Oligonucleotídeos/química , RNA/química , Rotaxanos/química , DNA/genética , Simulação de Acoplamento Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , RNA/genética , Termodinâmica
12.
Nucleic Acids Res ; 46(3): 1059-1068, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29309639

RESUMO

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.


Assuntos
Acridinas/química , Pareamento Incorreto de Bases , Replicação do DNA , DNA/química , Timidina/química , Triazinas/química , Compostos de Vinila/química , Alquilação , Pareamento de Bases , Sequência de Bases , DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Humanos , Terapia de Alvo Molecular , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Distrofia Miotônica/terapia , Timidina/metabolismo , Transcrição Gênica , Repetições de Trinucleotídeos
13.
Nucleic Acids Res ; 45(9): 5036-5047, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28407122

RESUMO

Circular nucleic acids have been utilized for versatile applications by taking advantage of the unique characteristic of their circular structure. In our previous study, we found that the chemically-cyclized ODN (cyODN) with double-tailed parts formed a pseudorotaxane structure with the target via the slippage process. We now report the investigation of the slippage properties and the mechanism of the slippage process using six different cyODNs. Our results indicate that the formation efficiency significantly depend on the temperature, the ring size, the target length and the mismatched position of the target. The kinetic studies also showed that this pseudorotaxane formation would proceed via a non-threaded structure which hybridizes with the target at the double-tailed parts. In addition, the resulting pseudorotaxanes showed interesting characteristics unlike the canonical duplex such as the hysteresis loop in the Tm measurements and the kinetic stabilization by lengthening the target. This information will be fundamentally important for finding new functions of circular nucleic acids and elucidating the threading mechanism regarding other synthetic small molecules and biopolymers.


Assuntos
Oligonucleotídeos/química , Rotaxanos/síntese química , Conformação de Ácido Nucleico
14.
Org Biomol Chem ; 16(9): 1436-1441, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29412214

RESUMO

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.


Assuntos
DNA/química , Quadruplex G , Alquilação , Sequência de Bases , DNA/genética , Telômero/genética
15.
Bioorg Med Chem ; 26(12): 3551-3558, 2018 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-29807700

RESUMO

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.


Assuntos
Acridinas/química , Alquilantes/química , Quadruplex G , Triazinas/química , Alquilação , Pareamento Incorreto de Bases , Sequência de Bases , Timina/química , Timina/metabolismo , Temperatura de Transição
16.
Bioorg Med Chem ; 25(7): 2191-2199, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28268052

RESUMO

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.


Assuntos
Conformação de Ácido Nucleico , RNA de Cadeia Dupla/síntese química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , RNA de Cadeia Dupla/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Am Chem Soc ; 138(28): 8667-9, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27387838

RESUMO

Short interfering RNAs (siRNAs) are promising therapeutics that make use of the RNA interference (RNAi) pathway, but liabilities arising from the native RNA structure necessitate chemical modification for drug development. Advances in the structural characterization of components of the human RNAi pathway have enabled structure-guided optimization of siRNA properties. Here we report the 2.3 Å resolution crystal structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi pathway, bound to an siRNA guide strand bearing an unnatural triazolyl nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue inserts deeply into hAgo2's central RNA binding cleft and thus is able to modulate pairing between guide and target RNAs. The affinity of the hAgo2-siRNA complex for a seed-only matched target was significantly reduced by the triazolyl modification, while the affinity for a fully matched target was unchanged. In addition, siRNA potency for off-target repression was reduced (4-fold increase in IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). Controlling siRNA on-target versus microRNA (miRNA)-like off-target potency by projection of substituent groups into the hAgo2 central cleft from g1 is a new approach to enhance siRNA selectivity with a strong structural rationale.


Assuntos
RNA Interferente Pequeno/genética , Proteínas Argonautas/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/deficiência , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Nucleotídeos/química , Interferência de RNA , RNA Interferente Pequeno/química , Triazóis/química
18.
Chembiochem ; 17(24): 2340-2345, 2016 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-27731539

RESUMO

Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Região 3'-Flanqueadora , Alcinos/química , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Azidas/química , Catálise , Classe I de Fosfatidilinositol 3-Quinases , Cobre/química , Reação de Cicloadição , Células HeLa , Humanos , MicroRNAs/química , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Domínios Proteicos , RNA Interferente Pequeno/metabolismo
19.
Nucleic Acids Res ; 42(13): 8808-15, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24957600

RESUMO

Modified nucleosides in natural RNA molecules are essential for their functions. Non-natural nucleoside analogues have been introduced into RNA to manipulate its structure and function. We have recently developed a new strategy for the in situ modification of RNA based on the functionality transfer reaction between an oligodeoxynucleotide probe and an RNA substrate. 2'-Deoxy-6-thioguanosine (6-thio-dG) was used as the platform to anchor the transfer group. In this study, a pyridinyl vinyl ketone moiety was newly designed as the transfer group with the expectation that a metal cation would form a chelate complex with the pyridinyl-2-keto group. It was demonstrated that the (E)-pyridinyl vinyl keto group was efficiently and specifically transferred to the 4-amino group of the opposing cytosine in RNA in the presence of NiCl2 with more than 200-fold accelerated rate compared with the previous system with the use of the diketo transfer group. Detailed mechanistic studies suggested that NiCl2 forms a bridging complex between the pyridinyl keto moiety and the N7 of the purine residue neighboring the cytosine residue of the RNA substrate to bring the groups in close proximity.


Assuntos
Citosina/química , DNA/química , RNA/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Níquel/química , Sondas de Oligonucleotídeos/química , Tionucleosídeos/química
20.
Chem Pharm Bull (Tokyo) ; 64(9): 1315-20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581636

RESUMO

Cross-linking is a widely-used technology in the studies of DNA, RNA and their complexes with proteins. Intrinsically active alkylating moieties and photo-activated agents are chemically or enzymatically incorporated into nucleic acids. Thionucleobases resemble the corresponding natural bases, and form cross-links by UVA irradiation. They form cross-links only with a site in close contact, thereby allowing identification of the contacts within the nucleic acids and/or between the nucleic acids and proteins in complex nucleoprotein assemblies. On the other hand, the thionucleobase forms a cross-link less efficiently for the reaction with the opposite natural base in the DNA duplex. In this study, 6-thioguanine was connected to 2'-deoxyribose through an ethylene linker at the 1'-position (Et-thioG). The linker was expected to bring the 6-thio group close to the nucleobase in the opposite strand. In a duplex in which the 2'-deoxy-6-thioguanosine (6-thio-dG) did not form a crosslink, Et-thioG efficiently formed crosslink with a high selectivity for T by UVA irradiation, but with a much lower efficiency for dA, dG, dC, 5-methyl-dC or dU. Interestingly, the yield of the photo-crosslinked product with dT was effectively improved in the presence of dithiothreitol or sodium hydrosulfide (NaSH) at a low UVA irradiation dose. The efficient and selective cross-link formation at a low UVA dose may be beneficial for the biological application of Et-thioG.


Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Desoxirribose/química , Etilenos/química , Tioguanina/química , Timidina/química , Reagentes de Ligações Cruzadas/síntese química , Processos Fotoquímicos , Raios Ultravioleta
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