Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA.

The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods.

Impact of bPNA Backbone Structural Constraints and Composition on Triplex Hybridization with DNA.

We report herein a study on the impact of bifacial peptide nucleic acid (bPNA) amino acid composition and backbone modification on DNA binding. A series of bPNA backbone variants with identical net charge were synthesized to display either 4 or 6 melamine (M) bases. These bases form thymine-melamine-thymine (TMT) base-triples, resulting in triplex hybrid stem structures with T-rich DNAs. Analyses of 6 M bPNA-DNA hybrids suggested that hybrid stability was linked to amino acid secondary structure propensities, prompting a more detailed study in shorter 4 M bPNAs. We synthesized 4 M bPNAs predisposed to adopt helical secondary structure via helix-turn nucleation in 7-residue bPNAs using double-click covalent stapling. Generally, hybrid stability improved upon stapling, but amino acid composition had a more significant effect. We also pursued an alternative strategy for bPNA structural preorganization by incorporation of residues with strong backbone amide conformational preferences such as 4R- and 4S-fluoroprolines. Notably, these derivatives exhibited an additional improvement in hybrid stability beyond both unsubstituted proline bPNA analogues and the helically patterned bPNAs. Overall, these findings demonstrate the tunability of bPNA-DNA hybrid stability through bPNA backbone structural propensities and amino acid composition.

Enhanced Triplex Hybridization of DNA and RNA via Syndiotactic Side Chain Presentation in Minimal bPNAs.

General design principles for recognition at noncanonical interfaces of DNA and RNA remain elusive. Triplex hybridization of bifacial peptide nucleic acids (bPNAs) with oligo-T/U DNAs and RNAs is a robust recognition platform that can be used to define structure-function relationships in synthetic triplex formation. To this end, a set of minimal (mw < 1 kD) bPNA variants was synthesized to probe the impact of amino acid secondary structural propensity, stereochemistry, and backbone cyclization on hybridization with short, unstructured T-rich DNA and U-rich RNAs. Thermodynamic parameters extracted from optical melting analyses of bPNA variant hybrids indicated that there are two bPNA backbone modifications that significantly improve hybridization: alternating (d, l) configuration in open-chain dipeptides and homochiral dipeptide cyclization to diketopiperazine. Further, binding to DNA is preferred over RNA for all bPNA variants. Thymine-uracil substitutions in DNA substrates revealed that the methyl group of thymine accounts for 71% of ΔΔGDNA-RNA for open-chain bPNAs but only 40% of ΔΔGDNA-RNA for diketopiperazine bPNA, suggesting a greater sensitivity to RNA conformation and more optimized stacking in the cyclic bPNA. Together, these data reveal pressure points for tuning triplex hybridization at the chiral centers of bPNA, backbone conformation, stacking effects at the base triple, and the nucleic acid substrate itself. A structural blueprint for enhancing bPNA targeting of both DNA and RNA substrates includes syndiotactic base presentation (as found in homochiral diketopiperazines and d, l peptides), expansion of base stacking, and further investigation of bPNA backbone preorganization.

Bifacial PNAs Destabilize MALAT1 by 3' A-Tail Displacement from the U-Rich Internal Loop.

We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3' oligo-A tail of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs composed of α-linked peptide, isodipeptide, and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE half-life plunging to 6 min from >24 h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinities, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 > 4000× , supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for the investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.

Unnatural bases for recognition of noncoding nucleic acid interfaces.

The notion of using synthetic heterocycles instead of the native bases to interface with DNA and RNA has been explored for nearly 60 years. Unnatural bases compatible with the DNA/RNA coding interface have the potential to expand the genetic code and co-opt the machinery of biology to access new macromolecular function; accordingly, this body of research is core to synthetic biology. While much of the literature on artificial bases focuses on code expansion, there is a significant and growing effort on docking synthetic heterocycles to noncoding nucleic acid interfaces; this approach seeks to illuminate major processes of nucleic acids, including regulation of transcription, translation, transport, and transcript lifetimes. These major avenues of research at the coding and noncoding interfaces have in common fundamental principles in molecular recognition. Herein, we provide an overview of foundational literature in biophysics of base recognition and unnatural bases in coding to provide context for the developing area of targeting noncoding nucleic acid interfaces with synthetic bases, with a focus on systems developed through iterative design and biophysical study.

Ferrier Carbocyclization-Mediated Synthesis of Enantiopure Azido Inositol Analogues.

Azide-modified inositol (InoAz) analogues are valuable as inhibitors and have shown promise as metabolic chemical reporters (MCRs) for labeling inositol-containing glycoconjugates in eukaryotic cells and potentially in mycobacteria, but the synthesis of enantiomerically pure InoAz analogues via traditional approaches is challenging. As a complementary route, here we investigated the application of the Ferrier carbocyclization reaction to the synthesis of enantiopure InoAz analogues starting from readily available azido glucosides. Using this approach combined with a para-methoxybenzyl protecting group strategy, 3-azido-3-deoxy- and 4-azido-4-deoxy-d-myo-inositol were efficiently synthesized. 5-Azido-5-deoxy-d-myo-inositol was inaccessible due to an unusual ß-elimination reaction, wherein the azide anion acted as the leaving group. The reported strategy is expected to facilitate continued development of synthetic InoAz analogues as inhibitors or MCRs of inositol-containing glycoconjugates in eukaryotic and mycobacterial systems.

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Deoxyfluoro-d-trehalose (FDTre) analogues as potential PET probes for imaging mycobacterial infection.

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, requires the non-mammalian disaccharide trehalose for growth and virulence. Recently, detectable trehalose analogues have gained attention as probes for studying trehalose metabolism and as potential diagnostic imaging agents for mycobacterial infections. Of particular interest are deoxy-[(18)F]fluoro-d-trehalose ((18)F-FDTre) analogues, which have been suggested as possible positron emission tomography (PET) probes for in vivo imaging of M. tuberculosis infection. Here, we report progress toward this objective, including the synthesis and conformational analysis of four non-radioactive deoxy-[(19)F]fluoro-d-trehalose ((19)F-FDTre) analogues, as well as evaluation of their uptake by M. smegmatis. The rapid synthesis and purification of several (19)F-FDTre analogues was accomplished in high yield using a one-step chemoenzymatic method. Conformational analysis of the (19)F-FDTre analogues using NMR and molecular modeling methods showed that fluorine substitution had a negligible effect on the conformation of the native disaccharide, suggesting that fluorinated analogues may be successfully recognized and processed by trehalose metabolic machinery in mycobacteria. To test this hypothesis and to evaluate a possible route for delivery of FDTre probes specifically to mycobacteria, we showed that (19)F-FDTre analogues are actively imported into M. smegmatis via the trehalose-specific transporter SugABC-LpqY. Finally, to demonstrate the applicability of these results to the efficient preparation and use of short-lived (18)F-FDTre PET radiotracers, we carried out (19)F-FDTre synthesis, purification, and administration to M. smegmatis in 1 hour.

Bioorthogonal Chemical Reporters for Selective In†Situ Probing of Mycomembrane Components in Mycobacteria.

The global pathogen Mycobacterium tuberculosis and other species in the suborder Corynebacterineae possess a distinctive outer membrane called the mycomembrane (MM). The MM is composed of mycolic acids, which are either covalently linked to an underlying arabinogalactan layer or incorporated into trehalose glycolipids that associate with the MM non-covalently. These structures are generated through a process called mycolylation, which is central to mycobacterial physiology and pathogenesis and is an important target for tuberculosis drug development. Current approaches to investigating mycolylation rely on arduous analytical methods that occur outside the context of a whole cell. Herein, we describe mycobacteria-specific chemical reporters that can selectively probe either covalent arabinogalactan mycolates or non-covalent trehalose mycolates in live mycobacteria. These probes, in conjunction with bioorthogonal chemistry, enable selective in situ detection of the major MM components.

A trifunctional cyclooctyne for modifying azide-labeled biomolecules with photocrosslinking and affinity tags.

A bicyclo[6.1.0]nonyne (BCN)-based cyclooctyne reagent bearing a photocrosslinking diazirine (DAz) group and a biotin affinity handle, BCN-DAz-Biotin, is reported. BCN-DAz-Biotin is capable of simultaneously delivering photocrosslinking and affinity tags to azide-labeled biomolecules, enabling photoactivated capture and enrichment/detection of interacting species in native contexts.