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Oligonucleotides as targets and cellular probes
Bioorganic & Medicinal Chemistry 21 (2013) 6099–6100
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Oligonucleotides as targets and cellular probes
Oligonucleotides are macromolecules with a wide range of biological function and are involved in many fundamental cellular processes, most importantly as the cellular storage material for genetic information (DNA). In addition, oligonucleotides also function as messenger RNAs (mRNAs), transfer RNAs (tRNAs), and various other non-coding RNAs (microRNAs, siRNAs, snoRNAs, etc). Oligonucleotides undergo selective hybridization to their complement sequences based on Watson-Crick base-pairing. This sequence-specific hybridization is highly programmable, based on hydrogen-bond interactions between A:T, A:U, and G:C base pairs. In addition to occurrence as single strands, duplexes, and triplexes, oligonucleotides can also form higher order structures by folding into G-quadruplexes or aptamers. These complex structures can be bound by small molecules, proteins, and other cellular components. Due to the biological importance of oligonucleotides, their programmability in sequence and function, as well as their potential for complex structure formation, oligonucleotides have been extensively targeted in therapeutic contexts and have been used as probes to investigate and study biological phenomena. This special issue presents selected topics approaching the chemistry and biology of oligonucleotides as targets and probes in exciting ways to help understand fundamental molecular mechanisms in Nature with the ultimate goal of improving human health. The first part of this collection of research articles and review articles focuses on the targeting of oligonucleotides by small synthetic molecules. The first review article (Melander) discusses recent applications of pyrrole–imidazole polyamides in the targeting of double-stranded DNA, as well as the historical context of these unique reagents. This is followed by two reviews (Arenz and Jin) that summarize the targeting and inhibition of the microRNA and RNA interference pathway with oligonucleotides and small molecules in vitro and in vivo. The discussed molecular probes have potential as important tools in the investigation of small RNA function, regulation, and biogenesis. A subsequent research paper by Zhang describes the discovery of a new microRNA small molecule inhibitor. The identified small molecules are interesting photo-cycloaddition products from reactions of acetylenes with naphtalenequinones and show good specificity and activity in tissue culture. Direct RNA–small molecule interactions were studied by Disney. Specifically, bacterial rRNA targeting with aminoglycosides was used as a starting point in investigations by a two-dimensional combinatorial screening platform, leading to the identification of modified aminoglycosides that bind new RNA motifs. The next two research articles discuss two success stories in targeting viral RNA for the development of new therapeutic 0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmc.2013.09.012
agents against hepatitis B and C virus infection. Here, Herman reports the development of a FRET-based assay for small molecule inhibitors of the highly conserved HCV IRES sequence. A pilot screen revealed a benzoxazole compound that effectively inhibits HCV translation in vitro. The following article by Engels describes the recruitment of the RNA interference pathway in the targeting and degradation of HCB RNA. In an extensive study, the authors found that activity and stability of the applied siRNA could be significantly improved through the site-specific incorporation of 20 -Oguanidinopropyl modifications, enabling efficient knock-down of viral replication in mice. In addition to double-stranded DNA, microRNAs, bacterial RNA, and viral RNA, G-quadruplex DNA is another important oligonucleotide motif that has been targeted with small molecule probes. In this context, Hartig provides a perspective on EPR approaches toward the investigation of G-quadruplex DNA conformations through site-specific introduction of a spinlabel modified nucleotide into the oligomer. Initial studies of applying this methodology to the study of G-quadruplex formation in live cells show substantial promise. Studies of G-quadruplex structures are followed by an article by Neidle, reporting the synthesis and biological investigation of tetra-substituted naphthalenediimides as selective G-quadruplex targeting compounds. Side-chain modifications of these small molecules led to potent quadruplex stabilizers that efficiently inhibited proliferation of a pancreatic cancer cell line. In the second part of this issue, oligonucleotides are being used as probes to manipulate biological processes. Höbartner reports a new method of introducing a spin-label into RNA for structural studies by EPR. The approach is based on the application of a convertible (4-chlorophenyl)uridine nucleotide in conjunction with ligation by a deoxyribozyme, generating spin-labeled sequences of up to 118 nucleotides. The next article describes an endonuclease-based detection method for both RNA and DNA, enabling isothermal sequence-specific quantification of oligonucleotides (Sintim). The applied junction probe design allows for detection with low background and does not require a specific oligonucleotide isolation step in sample preparation. Another detection method is reported that utilizes a quencher-free molecular beacon probe based on pyrene excimer fluorescence (Wengel). The potential of these probes for diagnostic purposes in the detection of RNA and DNA sequences was not only demonstrated in a test tube, but also in tissue culture. Further studies of chromophores incorporated into duplex DNA were discussed by Asanuma and Kashida, who reported a detailed investigation of insulating fluorophores from neighboring base pairs through site-specific positioning of
6100
Editorial / Bioorganic & Medicinal Chemistry 21 (2013) 6099–6100
cyclohexyl nucleotides. The resulting increase in fluorescence has potential implications in the design of new molecular beacon probes. This is followed by two reports on the application of light-removable protecting groups, so called caging groups, on oligonucleotide probes thereby enabling precise control over biological activity and bioconjugation reactions. Dmochowski presents an efficient approach to the photochemical control of microRNA function by modifying the microRNA sequence itself with caging groups or by inserting light-responsive groups into a microRNAinhibiting antagomir. The design of these oligonucleotides allowed for the light-activation and -deactivation of the let-7 microRNA in zebrafish embryos. The Tang group reports the enzymatic incorporation of nucleotide triphosphates that were modified with a caged carboxy or amino group into DNA. UV irradiation and decaging enabled temporal control of the bioconjugation of fluorophores to the oligonucleotides for site-specific dual labeling. The subsequent article describes an in vitro study investigating the effects of different hydrogen bonding patterns on modified nucleobases to pro-mutagenic DNA alkylation products (Sturla); investigations that may lead to better probes for the detection of nucleotide alkylation adducts. Delivering oligonucleotides into cells and nuclei remains an important biological problem that needs to be addressed before these reagents can be developed into targeted therapeutics. A potential solution could be the generation of ligand-oligonucleotide conjugates that facilitate receptormediated delivery into cells. The Juliano lab summarizes several
of these approaches that have been reported in the literature and provide their own unique insight regarding their advantages and disadvantages. This is followed by an article reporting the enhancement of cellular oligonucleotide uptake through incorporation of positively charged 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions that are thermally cleaved in cells, forming bioactive oligonucleotide phosphorothioate diesters (Beaucage). The last two publications in this special issue involve the application of RNA aptamers. The first report describes a new method of sensing cellular RNA expression by applying an aptamer targeting the bacterial tetracycline repressor together with a suitable reporter gene under control of the tetracycline operator (Süß). The second and final article reports the selection of aptamers for the guanine nucleotide exchange factor Tiam1, an important regulator of the Rho GTPase Rac that is involved in the development and progression of cancer (Famulok). The evolved aptamers selectively target the GTPase binding site of Tiam1 and thus effectively inhibit its function, thereby constituting new probes for the investigation of Tiam1 function and the development of new anticancer agents. Alexander Deiters University of Pittsburgh, Department of Chemistry, Pittsburgh, PA 15260, United States E-mail address: [email protected]
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Oligonucleotides as targets and cellular probes
Oligonucleotides are macromolecules with a wide range of biological function and are involved in many fundamental cellular processes, most importantly as the cellular storage material for genetic information (DNA). In addition, oligonucleotides also function as messenger RNAs (mRNAs), transfer RNAs (tRNAs), and various other non-coding RNAs (microRNAs, siRNAs, snoRNAs, etc). Oligonucleotides undergo selective hybridization to their complement sequences based on Watson-Crick base-pairing. This sequence-specific hybridization is highly programmable, based on hydrogen-bond interactions between A:T, A:U, and G:C base pairs. In addition to occurrence as single strands, duplexes, and triplexes, oligonucleotides can also form higher order structures by folding into G-quadruplexes or aptamers. These complex structures can be bound by small molecules, proteins, and other cellular components. Due to the biological importance of oligonucleotides, their programmability in sequence and function, as well as their potential for complex structure formation, oligonucleotides have been extensively targeted in therapeutic contexts and have been used as probes to investigate and study biological phenomena. This special issue presents selected topics approaching the chemistry and biology of oligonucleotides as targets and probes in exciting ways to help understand fundamental molecular mechanisms in Nature with the ultimate goal of improving human health. The first part of this collection of research articles and review articles focuses on the targeting of oligonucleotides by small synthetic molecules. The first review article (Melander) discusses recent applications of pyrrole–imidazole polyamides in the targeting of double-stranded DNA, as well as the historical context of these unique reagents. This is followed by two reviews (Arenz and Jin) that summarize the targeting and inhibition of the microRNA and RNA interference pathway with oligonucleotides and small molecules in vitro and in vivo. The discussed molecular probes have potential as important tools in the investigation of small RNA function, regulation, and biogenesis. A subsequent research paper by Zhang describes the discovery of a new microRNA small molecule inhibitor. The identified small molecules are interesting photo-cycloaddition products from reactions of acetylenes with naphtalenequinones and show good specificity and activity in tissue culture. Direct RNA–small molecule interactions were studied by Disney. Specifically, bacterial rRNA targeting with aminoglycosides was used as a starting point in investigations by a two-dimensional combinatorial screening platform, leading to the identification of modified aminoglycosides that bind new RNA motifs. The next two research articles discuss two success stories in targeting viral RNA for the development of new therapeutic 0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmc.2013.09.012
agents against hepatitis B and C virus infection. Here, Herman reports the development of a FRET-based assay for small molecule inhibitors of the highly conserved HCV IRES sequence. A pilot screen revealed a benzoxazole compound that effectively inhibits HCV translation in vitro. The following article by Engels describes the recruitment of the RNA interference pathway in the targeting and degradation of HCB RNA. In an extensive study, the authors found that activity and stability of the applied siRNA could be significantly improved through the site-specific incorporation of 20 -Oguanidinopropyl modifications, enabling efficient knock-down of viral replication in mice. In addition to double-stranded DNA, microRNAs, bacterial RNA, and viral RNA, G-quadruplex DNA is another important oligonucleotide motif that has been targeted with small molecule probes. In this context, Hartig provides a perspective on EPR approaches toward the investigation of G-quadruplex DNA conformations through site-specific introduction of a spinlabel modified nucleotide into the oligomer. Initial studies of applying this methodology to the study of G-quadruplex formation in live cells show substantial promise. Studies of G-quadruplex structures are followed by an article by Neidle, reporting the synthesis and biological investigation of tetra-substituted naphthalenediimides as selective G-quadruplex targeting compounds. Side-chain modifications of these small molecules led to potent quadruplex stabilizers that efficiently inhibited proliferation of a pancreatic cancer cell line. In the second part of this issue, oligonucleotides are being used as probes to manipulate biological processes. Höbartner reports a new method of introducing a spin-label into RNA for structural studies by EPR. The approach is based on the application of a convertible (4-chlorophenyl)uridine nucleotide in conjunction with ligation by a deoxyribozyme, generating spin-labeled sequences of up to 118 nucleotides. The next article describes an endonuclease-based detection method for both RNA and DNA, enabling isothermal sequence-specific quantification of oligonucleotides (Sintim). The applied junction probe design allows for detection with low background and does not require a specific oligonucleotide isolation step in sample preparation. Another detection method is reported that utilizes a quencher-free molecular beacon probe based on pyrene excimer fluorescence (Wengel). The potential of these probes for diagnostic purposes in the detection of RNA and DNA sequences was not only demonstrated in a test tube, but also in tissue culture. Further studies of chromophores incorporated into duplex DNA were discussed by Asanuma and Kashida, who reported a detailed investigation of insulating fluorophores from neighboring base pairs through site-specific positioning of
6100
Editorial / Bioorganic & Medicinal Chemistry 21 (2013) 6099–6100
cyclohexyl nucleotides. The resulting increase in fluorescence has potential implications in the design of new molecular beacon probes. This is followed by two reports on the application of light-removable protecting groups, so called caging groups, on oligonucleotide probes thereby enabling precise control over biological activity and bioconjugation reactions. Dmochowski presents an efficient approach to the photochemical control of microRNA function by modifying the microRNA sequence itself with caging groups or by inserting light-responsive groups into a microRNAinhibiting antagomir. The design of these oligonucleotides allowed for the light-activation and -deactivation of the let-7 microRNA in zebrafish embryos. The Tang group reports the enzymatic incorporation of nucleotide triphosphates that were modified with a caged carboxy or amino group into DNA. UV irradiation and decaging enabled temporal control of the bioconjugation of fluorophores to the oligonucleotides for site-specific dual labeling. The subsequent article describes an in vitro study investigating the effects of different hydrogen bonding patterns on modified nucleobases to pro-mutagenic DNA alkylation products (Sturla); investigations that may lead to better probes for the detection of nucleotide alkylation adducts. Delivering oligonucleotides into cells and nuclei remains an important biological problem that needs to be addressed before these reagents can be developed into targeted therapeutics. A potential solution could be the generation of ligand-oligonucleotide conjugates that facilitate receptormediated delivery into cells. The Juliano lab summarizes several
of these approaches that have been reported in the literature and provide their own unique insight regarding their advantages and disadvantages. This is followed by an article reporting the enhancement of cellular oligonucleotide uptake through incorporation of positively charged 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions that are thermally cleaved in cells, forming bioactive oligonucleotide phosphorothioate diesters (Beaucage). The last two publications in this special issue involve the application of RNA aptamers. The first report describes a new method of sensing cellular RNA expression by applying an aptamer targeting the bacterial tetracycline repressor together with a suitable reporter gene under control of the tetracycline operator (Süß). The second and final article reports the selection of aptamers for the guanine nucleotide exchange factor Tiam1, an important regulator of the Rho GTPase Rac that is involved in the development and progression of cancer (Famulok). The evolved aptamers selectively target the GTPase binding site of Tiam1 and thus effectively inhibit its function, thereby constituting new probes for the investigation of Tiam1 function and the development of new anticancer agents. Alexander Deiters University of Pittsburgh, Department of Chemistry, Pittsburgh, PA 15260, United States E-mail address: [email protected]