Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA.

Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called "one-label, one-technique" approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and 19F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2'-deoxyuridine, which serves as an excellent fluorescent and 19F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and 19F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and 19F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.

Probing the Competition between Duplex, G-Quadruplex and i-Motif Structures of the Oncogenic c-Myc DNA Promoter Region.

Development of probe systems that provide unique spectral signatures for duplex, G-quadruplex (GQ) and i-motif (iM) structures is very important to understand the relative propensity of a G-rich-C-rich promoter region to form these structures. Here, we devise a platform using a combination of two environment-sensitive nucleoside analogs namely, 5-fluorobenzofuran-modified 2'-deoxyuridine (FBF-dU) and 5-fluoro-2'-deoxyuridine (F-dU) to study the structures adopted by a promoter region of the c-Myc oncogene. FBF-dU serves as a dual-purpose probe containing a fluorescent and 19 F NMR label. When incorporated into the C-rich sequence, it reports the formation of different iMs via changes in its fluorescence properties and 19 F signal. F-dU incorporated into the G-rich ON reports the formation of a GQ structure whose 19 F signal is clearly different from the signals obtained for iMs. Rewardingly, the labeled ONs when mixed with respective complementary strands allows us to determine the relative population of different structures formed by the c-Myc promoter by the virtue of the probe's ability to produce distinct and resolved 19 F signatures for different structures. Our results indicate that at physiological pH and temperature the c-Myc promoter forms duplex, random coil and GQ structures, and does not form an iM. Whereas at acidic pH, the mixture largely forms iM and GQ structures. Taken together, our system will complement existing tools and provide unprecedented insights on the population equilibrium and dynamics of nucleic acid structures under different conditions.

Probing juxtaposed G-quadruplex and hairpin motifs using a responsive nucleoside probe: a unique scaffold for chemotherapy.

Paucity of efficient probes and small molecule ligands that can distinguish different G-quadruplex (GQ) topologies poses challenges not only in understanding their basic structure but also in targeting an individual GQ form from others. Alternatively, G-rich sequences that harbour unique chimeric structural motifs (e.g., GQ-duplex or GQ-hairpin junctions) are perceived as new therapeutic hotspots. In this context, the epidermal growth factor receptor (EGFR) gene, implicated in many cancers, contains a 30 nucleotide G-rich segment in the promoter region, which adopts in vitro two unique architectures each composed of a GQ topology (parallel and hybrid-type) juxtaposed with a hairpin domain. Here, we report the use of a novel dual-app probe, C5-trifluoromethyl benzofuran-modified 2'-deoxyuridine (TFBF-dU), in the systematic analysis of EGFR GQs and their interaction with small molecules by fluorescence and 19F NMR techniques. Notably, distinct fluorescence and 19F NMR signals exhibited by the probe enabled the quantification of the relative population of random, parallel and hybrid-type GQ structures under different conditions, which could not be obtained by conventional CD and 1H NMR techniques. Using the fluorescence component, we quantified ligand binding properties of GQs, whereas the 19F label enabled the assessment of ligand-induced changes in GQ dynamics. Studies also revealed that mutations in the hairpin domain affected GQ formation and stability, which was further functionally verified in polymerase stop assay. We anticipate that these findings and useful properties of the nucleoside probe could be utilized in designing and evaluating binders that jointly target both GQ and hairpin domains for enhanced selectivity and druggability.

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.

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.

Heterocycle-modified 2'-Deoxyguanosine Nucleolipid Analogs Stabilize Guanosine Gels and Self-assemble to Form Green Fluorescent Gels.

Nucleoside-lipid conjugates are very useful supramolecular building blocks to construct self-assembled architectures suited for biomedical and material applications. Such nucleoside derivatives can be further synthetically manipulated to endow additional functionalities that could augment the assembling process and impart interesting properties. Here, we report the design, synthesis and self-assembling process of multifunctional supramolecular nucleolipid synthons containing an environment-sensitive fluorescent guanine. The amphiphilic synthons are composed of an 8-(2-(benzofuran-2-yl)vinyl)-guanine core and alkyl chains attached to 3'-O and 5'-O-positions of 2'-deoxyguanosine. The 2-(benzofuran-2-yl)vinyl (BFV) moiety attached at the C8 position of the nucleobase adopted a syn conformation about the glycosidic bond, which facilitated the self-assembly process through the formation of a G-tetrad as the basic unit. While 3',5'-diacylated BFV-modified dG analog stabilized the guanosine hydrogel by hampering the crystallization process and imparted fluorescence, BFV-modified dGs containing longer alkyl chains formed a green fluorescent organogel, which transformed into a yellow fluorescent gel in the presence of a complementary non-fluorescent cytidine nucleolipid. The ability of the dG analog containing short alkyl chains to modulate the mechanical property of a gel, and interesting fluorescence properties and self-assembling behavior exhibited by the dG analogs containing long alkyl chains in response to heat and complementary base underscore the potential use of these new supramolecular synthons in material applications.

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.

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.

Bioorthogonal chemistry-based RNA labeling technologies: evolution and current state.

To understand the structure and ensuing function of RNA in various cellular processes, researchers greatly rely on traditional as well as contemporary labeling technologies to devise efficient biochemical and biophysical platforms. In this context, bioorthogonal chemistry based on chemoselective reactions that work under biologically benign conditions has emerged as a state-of-the-art labeling technology for functionalizing biopolymers. Implementation of this technology on sugar, protein, lipid and DNA is fairly well established. However, its use in labeling RNA has posed challenges due to the fragile nature of RNA. In this feature article, we provide an account of bioorthogonal chemistry-based RNA labeling techniques developed in our lab along with a detailed discussion on other technologies put forward recently. In particular, we focus on the development and applications of covalent methods to label RNA by transcription and posttranscription chemo-enzymatic approaches. It is expected that existing as well as new bioorthogonal functionalization methods will immensely advance our understanding of RNA and support the development of RNA-based diagnostic and therapeutic tools.

Responsive fluorescent nucleotides serve as efficient substrates to probe terminal uridylyl transferase.

We repurposed a terminal uridylyl transferase enzyme to site-specifically label RNA with microenvironment sensing fluorescent nucleotide mimics, which in turn provided direct read-outs to estimate the binding affinities of the enzyme to RNA and nucleotide substrates. This enzyme-probe system provides insights into the catalytic cycle, and can facilitate the development of discovery platforms to identify robust enzyme inhibitors.

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.

Posttranscriptional Suzuki-Miyaura Cross-Coupling Yields Labeled RNA for Conformational Analysis and Imaging.

Chemical labeling of RNA by using chemoselective reactions that work under biologically benign conditions is increasingly becoming valuable in the in vitro and in vivo analysis of RNA. Here, we describe a modular RNA labeling method based on a posttranscriptional Suzuki-Miyaura coupling reaction, which works under mild conditions and enables the direct installation of various biophysical reporters and tags. This two-part procedure involves the incorporation of a halogen-modified UTP analog (5-iodouridine-5'-triphosphate) by a transcription reaction. Subsequent posttranscriptional coupling with boronic acid/ester substrates in the presence of a palladium catalyst provides access to RNA labeled with (a) fluorogenic environment-sensitive nucleosides for probing nucleic acid structure and recognition, (b) fluorescent probes for microscopy, and (3) affinity tags for pull-down and immunoassays. It is expected that this method could also become useful for imaging nascent RNA transcripts in cells if the nucleotide analog can be metabolically incorporated and coupled with reporters by metal-assisted cross-coupling reactions.

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.

Synthesis of DNA and RNA Oligonucleotides Containing a Dual-Purpose Selenium-Modified Fluorescent Nucleoside Probe.

Development of efficient tools that would enable direct correlation of nucleic acid structure and recognition in solution and in solid state at atomic resolution is highly desired. In this context, we recently developed dual-purpose nucleoside probes made of a 5-selenophene-modified uracil core, which serves both as a conformation-sensitive fluorophore and as an X-ray crystallography phasing agent. In this article, we provide a detailed synthetic procedure to synthesize the phosphoramidites of 5-selenophene-modified 2'-deoxyuridine and 5-selenophene-modified uridine analogs. We also describe their site-specific incorporation into therapeutically relevant DNA and RNA oligonucleotide motifs by an automated solid support synthesis protocol. The dual-purpose and minimally invasive nature of the probes enables efficient analysis of the conformation and ligand binding abilities of bacterial decoding site RNA (A-site) and G-quadruplex structures of the human telomeric overhang in real time by fluorescence and in 3D by X-ray crystallography. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 5-selenophene-2'-deoxyuridine 2 and its phosphoramidite 5 Support Protocol 1: Synthesis of 2-(tri-n-butylstannyl) selenophene Support Protocol 2: Synthesis of 5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 Basic Protocol 2: Synthesis of 5-selenophene-modified uridine 7 and its phosphoramidite 11 Basic Protocol 3: Synthesis of DNA oligonucleotides containing 5-selenophene-modified 2'-deoxyuridine 2 Basic Protocol 4: Synthesis of an RNA oligonucleotide containing 5-selenophene-modified uridine 7.

Multi-stimuli responsive heterotypic hydrogels based on nucleolipids show selective dye adsorption.

Analogous to nucleic acids, the building blocks of nucleic acids and their derivatives are widely used to create supramolecular architectures for application mainly in the field of biomedicine. Here, we describe the construction of a multi-stimuli responsive and toxic dye adsorbing heterotypic hydrogel system formed using simple nucleoside-fatty acid conjugates. The nucleolipids are derived by coupling fatty acid chains of different lengths at the 5' position of ribothymidine and uridine. The nucleolipids in the presence of a strong base (e.g. NaOH) undergo partial hydrolysis, which triggers the self-assembly of the hydrolysed components resulting in the formation of heterotypic hydrogels. Notably, the gels are formed specifically in the presence of Na+ ions as other ions such as Li+ and K+ did not support the hydrogelation process. Systematic analysis by microscopy, NMR, single crystal and powder X-ray diffraction and rheology indicated that the deprotonated nucleolipid and fatty acid salt interdigitate and provide necessary electrostatic interactions supported by Na+ ions to set the path for the hierarchical assembly process. Notably, the hydrogels are highly sensitive to external stimuli, wherein gel-sol transition can be reversibly controlled by using temperature, pH and host-guest interaction. One of the hydrogels made of 5'-O-myristate-conjugated ribothymidine was found to selectively adsorb cationic dyes such as methylene blue and rhodamine 6G in a recyclable fashion. Taken together, the easily scalable assembly, multi-stimuli responsiveness and ability to capture and release dyes highlight the potential of our nucleolipid hydrogel system in material applications and in the treatment of dye industry wastes.

Self-assemblies of nucleolipid supramolecular synthons show unique self-sorting and cooperative assembling process.

The inherent control of the self-sorting and co-assembling process that has evolved in multi-component biological systems is not easy to emulate in vitro using synthetic supramolecular synthons. Here, using the basic component of nucleic acids and lipids, we describe a simple platform to build hierarchical assemblies of two component systems, which show an interesting self-sorting and co-assembling behavior. The assembling systems are made of a combination of amphiphilic purine and pyrimidine ribonucleoside-fatty acid conjugates (nucleolipids), which were prepared by coupling fatty acid acyl chains of different lengths at the 2'-O- and 3'-O-positions of the ribose sugar. Individually, the purine and pyrimidine nucleolipids adopt a distinct morphology, which either supports or does not support the gelation process. Interestingly, due to the subtle difference in the order of formation and stability of individual assemblies, different mixtures of supramolecular synthons and complementary ribonucleosides exhibit a cooperative and disruptive self-sorting and co-assembling behavior. A systematic morphological analysis combined with single crystal X-ray crystallography, powder X-ray diffraction (PXRD), NMR, CD, rheological and 3D X-ray microtomography studies provided insights into the mechanism of the self-sorting and co-assembling process. Taken together, this approach has enabled the construction of assemblies with unique higher ordered architectures and gels with remarkably enhanced mechanical strength that cannot be derived from the respective single component systems.

Synthesis and Enzymatic Incorporation of a Responsive Ribonucleoside Probe That Enables Quantitative Detection of Metallo-Base Pairs.

Synthesis of a highly responsive fluorescent ribonucleoside analogue based on a 5-methoxybenzofuran uracil core, enzymatic incorporation of its triphosphate substrate into RNA transcripts, and its utility in the specific detection and estimation of Hg2+-ion-mediated metallo-base pair formation in DNA-RNA and RNA-RNA duplexes are described.

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.

Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs.

The ability to bind strongly to complementary nucleic acid sequences, invade complex nucleic acid structures, and resist degradation by cellular enzymes has made peptide nucleic acid (PNA) oligomers as very useful hybridization probes in molecular diagnosis. For such applications, the PNA oligomers have to be labeled with appropriate reporters as they lack intrinsic labels that can be used in biophysical assays. Although solid-phase synthesis is commonly used to attach reporters onto PNA, development of milder and modular labeling methods will provide access to PNA oligomers labeled with a wider range of biophysical tags. Here, we describe the establishment of a postsynthetic modification strategy based on bioorthogonal chemical reactions in functionalizing PNA oligomers in solution with a variety of tags. A toolbox composed of alkyne- and azide-modified monomers were site-specifically incorporated into PNA oligomers and postsynthetically click-functionalized with various tags, ranging from sugar, amino acid, biotin, to fluorophores, by using copper(I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, and Staudinger ligation reactions. As a proof of utility of this method, fluorescent PNA hybridization probes were developed and used in imaging human telomeres in chromosomes and poly(A) RNAs in cells. Taken together, this simple approach of generating a wide range of functional PNA oligomers will expand the use of PNA in molecular diagnosis.