Microenvironment-Sensitive Fluorescent Nucleotide Probes from Benzofuran, Benzothiophene, and Selenophene as Substrates for DNA Polymerases.

DNA polymerases can process a wide variety of structurally diverse nucleotide substrates, but the molecular basis by which the analogs are processed is not completely understood. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of DNA polymerases in real time and at the atomic level. The nucleotide analogs containing a selenophene, benzofuran, or benzothiophene moiety at the C5 position of 2'-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. KlenTaq (A family DNA polymerase) is sensitive to the size of the modification as it incorporates only one heterobicycle-modified nucleotide into the growing polymer, whereas it efficiently incorporates the selenophene-modified nucleotide analog at multiple positions. Notably, in the single nucleotide incorporation assay, irrespective of the heterocycle size, it exclusively adds a single nucleotide at the 3'-end of a primer, which enabled devising a simple two-step site-specific ON labeling technique. KOD and Vent(exo-) DNA polymerases, belonging to the B family, tolerate all the three modified nucleotides and produce ONs with multiple labels. Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real-time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides.

Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19 F NMR Spectroscopy.

Development of versatile probes that can enable the study of different conformations and recognition properties of therapeutic nucleic acid motifs by complementing biophysical techniques can greatly aid nucleic acid analysis and therapy. Here, we report the design, synthesis and incorporation of an environment-sensitive ribonucleoside analogue, which serves as a two-channel biophysical platform to investigate RNA structure and recognition by fluorescence and 19 F NMR spectroscopy techniques. The nucleoside analogue is based on a 5-fluorobenzofuran-uracil core and its fluorescence and 19 F NMR chemical shifts are highly sensitive to changes in solvent polarity and viscosity. Notably, the modified ribonucleotide and phosphoramidite substrates can be efficiently incorporated into RNA oligonucleotides (ONs) by in vitro transcription and standard solid-phase ON synthesis protocol, respectively. Fluorescence and 19 F readouts of the nucleoside incorporated into model RNA ONs are sensitive to the neighbouring base environment. The responsiveness of the probe was aptly utilized in detecting and quantifying the metal ion-induced conformational change in an internal ribosome entry site RNA motif of hepatitis C virus, which is an important therapeutic target. Taken together, our probe is a good addition to the nucleic acid analysis toolbox with the advantage that it can be used to study nucleic acid conformation and recognition simultaneously by two biophysical techniques.

Environment-Sensitive Nucleoside Probe Unravels the Complex Structural Dynamics of i-Motif DNAs.

Although evidence for the existence and biological role of i-motif (iM) DNA structures in cells is emerging, probing their structural polymorphism and identifying physiologically active conformations using currently available tools remain a major challenge. Here, we describe the development of an innovative device to investigate the conformation equilibrium of different iMs formed by C-rich telomeric repeat and oncogenic B-raf promoter sequences using a new conformation-sensitive dual-purpose nucleoside probe. The nucleoside is composed of a trifluoromethyl-benzofuran-2-yl moiety at the C5 position of 2'-deoxyuridine, which functions as a responsive fluorescent and 19F NMR probe. While the fluorescent component is useful in monitoring and estimating the folding process, the 19F label provides spectral signatures for various iMs, thereby enabling a systematic analysis of their complex population equilibrium under different conditions (e.g., pH, temperature, metal ions, and cell lysate). Distinct 19F signals exhibited by the iMs formed by the human telomeric repeat helped in calculating their relative population. A battery of fluorescence and 19F NMR studies using native and mutated B-raf oligonucleotides gave valuable insights into the iM structure landscape and its dependence on environmental conditions and also helped in predicting the structure of the major iM conformation. Overall, our findings indicate that the probe is highly suitable for studying complex nucleic acid systems.

Probing G-quadruplex topologies and recognition concurrently in real time and 3D using a dual-app nucleoside probe.

Comprehensive understanding of structure and recognition properties of regulatory nucleic acid elements in real time and atomic level is highly important to devise efficient therapeutic strategies. Here, we report the establishment of an innovative biophysical platform using a dual-app nucleoside analog, which serves as a common probe to detect and correlate different GQ structures and ligand binding under equilibrium conditions and in 3D by fluorescence and X-ray crystallography techniques. The probe (SedU) is composed of a microenvironment-sensitive fluorophore and an excellent anomalous X-ray scatterer (Se), which is assembled by attaching a selenophene ring at 5-position of 2'-deoxyuridine. SedU incorporated into the loop region of human telomeric DNA repeat fluorescently distinguished subtle differences in GQ topologies and enabled quantify ligand binding to different topologies. Importantly, anomalous X-ray dispersion signal from Se could be used to determine the structure of GQs. As the probe is minimally perturbing, a direct comparison of fluorescence data and crystal structures provided structural insights on how the probe senses different GQ conformations without affecting the native fold. Taken together, our dual-app probe represents a new class of tool that opens up new experimental strategies to concurrently investigate nucleic acid structure and recognition in real time and 3D.

Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9.

Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.

Nucleic Acid Conformation Influences Postsynthetic Suzuki-Miyaura Labeling of Oligonucleotides.

Chemoselective transformations that work under physiological conditions have emerged as powerful tools to label nucleic acids in cell-free and cellular environments. However, detailed studies investigating the influence of nucleic acid conformation on the performance of such chemoselective nucleic labeling methods are less explored. Given that nucleic acids adopt complex structures, it is highly important to study the scope of the chemical modification method in the context of nucleic acid conformations. Here we report a systematic study on the effect of local conformation on the postsynthetic Suzuki-Miyaura functionalization of human telomeric (H-Telo) DNA repeat oligonucleotide (ON) sequences, which form multiple G-quadruplex (GQ) structures. 5-Iodo-2'-deoxyuridine (IdU)-modified H-Telo ONs were synthesized by the solid-phase method, and when subjected to Suzuki-Miyaura cross-coupling reaction, its efficiency was found to depend on the type of conformation and the position of IdU label in different loops of the GQ structure. IdU-labeled GQs gave better yields as compared to single-stranded random coil structures. However, the IdU-labeled duplex under different ionic conditions did not undergo the coupling reaction. Further, using this method, we directly installed an environment-sensitive fluorescent probe, which photophysically reported the formation as well as distinguished different GQ topologies of telomeric repeat. Collectively, this systematic study underscores the influence of nucleic acid conformation, which has to be taken into account when establishing postsynthetic chemoselective functionalization strategies.

A dual-app nucleoside probe reports G-quadruplex formation and ligand binding in the long terminal repeat of HIV-1 proviral genome.

We have developed a dual-app nucleoside analog, 5-selenophene-modified 2'-deoxyuridine (SedU), to probe the structure and ligand-binding properties of a G-rich segment present in the long terminal repeat (LTR) of the HIV-1 proviral DNA promoter region. The nucleoside probe is made of an environment-responsive fluorophore and X-ray crystallography phasing label (Se atom). SedU incorporated into LTR-IV sequence, fluorescently reports the formation of G-quadruplex (GQ) structure without affecting the native fold. Further, using the environment sensitivity of the probe, a fluorescence assay was designed to estimate the binding affinity of small molecule ligands to the GQ motif. An added feature of this probe system is that it would enable direct correlation of structure and recognition properties in solution and atomic level by using a combination of fluorescence and X-ray crystallography techniques.

Post-transcriptional labeling by using Suzuki-Miyaura cross-coupling generates functional RNA probes.

Pd-catalyzed C-C bond formation, an important vertebra in the spine of synthetic chemistry, is emerging as a valuable chemoselective transformation for post-synthetic functionalization of biomacromolecules. While methods are available for labeling protein and DNA, development of an analogous procedure to label RNA by cross-coupling reactions remains a major challenge. Herein, we describe a new Pd-mediated RNA oligonucleotide (ON) labeling method that involves post-transcriptional functionalization of iodouridine-labeled RNA transcripts by using Suzuki-Miyaura cross-coupling reaction. 5-Iodouridine triphosphate (IUTP) is efficiently incorporated into RNA ONs at one or more sites by T7 RNA polymerase. Further, using a catalytic system made of Pd(OAc)2 and 2-aminopyrimidine-4,6-diol (ADHP) or dimethylamino-substituted ADHP (DMADHP), we established a modular method to functionalize iodouridine-labeled RNA ONs in the presence of various boronic acid and ester substrates under very mild conditions (37°C and pH 8.5). This method is highly chemoselective, and offers direct access to RNA ONs labeled with commonly used fluorescent and affinity tags and new fluorogenic environment-sensitive nucleoside probes in a ligand-controlled stereoselective fashion. Taken together, this simple approach of generating functional RNA ON probes by Suzuki-Miyaura coupling will be a very important addition to the resources and tools available for analyzing RNA motifs.

A Dual-App Nucleoside Probe Provides Structural Insights into the Human Telomeric Overhang in Live Cells.

Understanding the topology adopted by individual G-quadruplex (GQ)-forming sequences in vivo and targeting a specific GQ motif among others in the genome will have a profound impact on GQ-directed therapeutic strategies. However, this remains a major challenge as most of the tools poorly distinguish different GQ conformations and are not suitable for both cell-free and in-cell analysis. Here, we describe an innovative probe design to investigate GQ conformations and recognition in both cell-free and native cellular environments by using a conformation-sensitive dual-app nucleoside analogue probe. The nucleoside probe, derived by conjugating fluorobenzofuran at the 5-position of 2'-deoxyuridine, is composed of a microenvironment-sensitive fluorophore and an in-cell NMR compatible 19F label. This noninvasive nucleoside, incorporated into the human telomeric DNA oligonucleotide repeat, serves as a common probe to distinguish different GQ topologies and quantify topology-specific binding of ligands by fluorescence and NMR techniques. Importantly, unique signatures displayed by the 19F-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. Studies using synthetic cell models, immunostaining on fixed cells, and crystallization conditions suggest that parallel GQ is the preferred conformation of telomeric DNA repeat. However, our findings using the dual-app probe clearly indicate that multiple structures including hybrid-type parallel-antiparallel and parallel GQs are formed in the cellular environment. Taken together, our findings open new experimental strategies to investigate topology, recognition, and therapeutic potential of individual GQ-forming motifs in a biologically relevant context.

A Lucifer-Based Environment-Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs.

Fluorescence-based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment-sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8-naphthalimide) at the 5-position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells-poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.

Probing the competition between duplex and G-quadruplex/i-motif structures using a conformation-sensitive fluorescent nucleoside probe.

Double-stranded segments of a genome that can potentially form G-quadruplex (GQ) and/or i-motif (iM) structures are considered to be important regulatory elements. Hence, the development of a common probe that can detect GQ and iM structures and also distinguish them from a duplex structure will be highly useful in understanding the propensity of such segments to adopt duplex or non-canonical four-stranded structures. Here, we describe the utility of a conformation-sensitive fluorescent nucleoside analog, which was originally developed as a GQ sensor, in detecting the iM structures of C-rich DNA oligonucleotides (ONs). The analog is based on a 5-(benzofuran-2-yl)uracil scaffold, which when incorporated into C-rich ONs (e.g., telomeric repeats) fluorescently distinguishes an iM from random coil and duplex structures. Steady-state and time-resolved fluorescence techniques enabled the determination of transition pH for the transformation of a random coil to an iM structure. Furthermore, a qualitative understanding on the relative population of duplex and GQ/iM forms under physiological conditions could be gained by correlating the fluorescence, CD and thermal melting data. Taken together, this sensor could provide a general platform to profile double-stranded promoter regions in terms of their ability to adopt four-stranded structures, and also could support approaches to discover functional GQ and iM binders.

Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry.

Recent developments in RNA labeling technology have provided viable tools to analyze RNA synthesis, processing and function in cell-free and cellular environments. Notably, emerging methodologies based on posttranscriptional chemical labeling by using bioorthogonal chemistry have enabled the visualization and profiling of exogenous and endogenous RNA transcripts. In this review, we first give an overview of different RNA labeling strategies based on chemical as well as genetically encoded systems. Subsequently, we provided a detailed discussion on methodologies that have been developed to introduce various bioorthogonal reactive groups into RNA transcripts, which are compatible for posttranscriptional functionalization. Finally, the utility of these techniques in imaging and studying the dynamics of RNA production, distribution and decay in complex cellular environment is discussed.

A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.

Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.

Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential.

Vinyluridine as a Versatile Chemoselective Handle for the Post-transcriptional Chemical Functionalization of RNA.

The development of modular and efficient methods to functionalize RNA with biophysical probes is very important in advancing the understanding of the structural and functional relevance of RNA in various cellular events. Herein, we demonstrate a two-step bioorthogonal chemical functionalization approach for the conjugation of multiple probes onto RNA transcripts using a 5-vinyl-modified uridine nucleotide analog (VUTP). VUTP, containing a structurally noninvasive and versatile chemoselective handle, was efficiently incorporated into RNA transcripts by in vitro transcription reactions. Furthermore, we show for the first time the use of a palladium-mediated oxidative Heck reaction in functionalizing RNA with fluorogenic probes by reacting vinyl-labeled RNA transcripts with appropriate boronic acid substrates. The vinyl label also permitted the post-transcriptional functionalization of RNA by a reagent-free inverse electron demand Diels-Alder (IEDDA) reaction in the presence of tetrazine substrates. Collectively, our results demonstrate that the incorporation of VUTP provides newer possibilities for the modular functionalization of RNA with variety of reporters.

Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes.

Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2'-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential.

Structure of the Ribosomal RNA Decoding Site Containing a Selenium-Modified Responsive Fluorescent Ribonucleoside Probe.

Comprehensive understanding of the structure-function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation-sensitive fluorophore and an anomalous X-ray diffraction label (5-selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand-induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

Responsive Fluorescent PNA Analogue as a Tool for Detecting G-quadruplex Motifs of Oncogenes and Activity of Toxic Ribosome-Inactivating Proteins.

Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic-acid-based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment-sensitive 5-(benzofuran-2-yl)- and 5-(benzothiophen-2-yl)-uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene-modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn-on detection of G-quadruplex-forming promoter DNA sequences of human proto-oncogenes (c-myc and c-kit). Furthermore, the ability of benzothiophene-modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome-inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.

A clickable UTP analog for the posttranscriptional chemical labeling and imaging of RNA.

The development of robust tools and practical RNA labeling strategies that would facilitate the biophysical analysis of RNA in both cell-free and cellular systems will have profound implications in the discovery of new RNA diagnostic tools and therapeutic strategies. In this context, we describe the development of a new alkyne-modified UTP analog, 5-(1,7-octadinyl)uridine triphosphate (ODUTP), which serves as an efficient substrate for the introduction of a clickable alkyne label into RNA transcripts by bacteriophage T7 RNA polymerase and mammalian cellular RNA polymerases. The ODU-labeled RNA is effectively used by reverse transcriptase to produce cDNA, a property which could be utilized in expanding the chemical space of a RNA library in the aptamer selection scheme. Further, the alkyne label on RNA provides a convenient tool for the posttranscriptional chemical functionalization with a variety of biophysical tags (fluorescent, affinity, amino acid and sugar) by using alkyne-azide cycloaddition reaction. Importantly, the ability of endogenous RNA polymerases to specifically incorporate ODUTP into cellular RNA transcripts enabled the visualization of newly transcribing RNA in cells by microscopy using click reactions. In addition to a clickable alkyne group, ODU contains a Raman scattering label (internal disubstituted alkyne), which exhibits characteristic Raman shifts that fall in the Raman-silent region of cells. Our results indicate that an ODU label could potentially facilitate two-channel visualization of RNA in cells by using click chemistry and Raman spectroscopy. Taken together, ODU represents a multipurpose ribonucleoside tool, which is expected to provide new avenues to study RNA in cell-free and cellular systems.