Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes.

HIV-1 Gag proteins can multimerize upon the viral genomic RNA or multiple random cellular messenger RNAs to form a virus particle or a virus-like particle, respectively. To date, whether the two types of particles form via the same Gag multimerization process has remained unclarified. Using photoactivated localization microscopy to illuminate Gag organizations and dynamics at the nanoscale, here, we showed that genomic RNA mediates Gag multimerization in a more cluster-centric, cooperative, and spatiotemporally coordinated fashion, with the ability to drive dense Gag clustering dependent on its ability to act as a long-stranded scaffold not easily attainable by cellular messenger RNAs.

Spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium.

Molecular knowledge of human gastric corpus epithelium remains incomplete. Here, by integrated analyses using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) techniques, we uncovered the spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium. Specifically, we identified a stem/progenitor cell population in the isthmus of human gastric corpus, where EGF and WNT signaling pathways were activated.

MyoD is a 3D genome structure organizer for muscle cell identity.

The genome exists as an organized, three-dimensional (3D) dynamic architecture, and each cell type has a unique 3D genome organization that determines its cell identity. An unresolved question is how cell type-specific 3D genome structures are established during development. Here, we analyzed 3D genome structures in muscle cells from mice lacking the muscle lineage transcription factor (TF), MyoD, versus wild-type mice.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem-loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions.

A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein-Protein Interactions.

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein-protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. While it is thought that expressing the fusion proteins at a low level can remove false positives without impacting specific signals, there has been no effective strategy to test this possibility. Here, we present a system capable of assessing and removing BiFC false positives, termed Background Assessable and Correctable-BiFC (BAC-BiFC), in which one of the split FP fragments is fused with an optically distinct FP that serves as a reference marker, and the single-cell fluorescence ratio of the BiFC signal to the reference signal is used to gauge an optimal transfection condition.

1/f-noise-free optical sensing with an integrated heterodyne interferometer.

Optical evanescent sensors can non-invasively detect unlabeled nanoscale objects in real time with unprecedented sensitivity, enabling a variety of advances in fundamental physics and biological applications. However, the intrinsic low-frequency noise therein with an approximately 1/f-shaped spectral density imposes an ultimate detection limit for monitoring many paramount processes, such as antigen-antibody reactions, cell motions and DNA hybridizations. Here, we propose and demonstrate a 1/f-noise-free optical sensor through an up-converted detection system.

Recent Advances in the Molecular Beacon Technology for Live-Cell Single-Molecule Imaging.

Nucleic acids, aside from being best known as the carrier of genetic information, are versatile biomaterials for constructing nanoscopic devices for biointerfacing, owing to their unique properties such as specific base pairing and predictable structure. For live-cell analysis of native RNA transcripts, the most widely used nucleic acid-based nanodevice has been the molecular beacon (MB), a class of stem-loop-forming probes that is activated to fluoresce upon hybridization with target RNA. Here, we overview efforts that have been made in developing MB-based bioassays for sensitive intracellular analysis, particularly at the single-molecule level.

Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems.

The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein 9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the sgRNA with molecular beacons, a class of small molecule dye-based, fluorogenic oligonucleotide probes, and demonstrated that the resulting system, termed CRISPR/MB, could be more sensitive and quantitative than conventional approaches employing FP reporters in detecting single telomere loci.

A new metagenome binning method based on gene uniqueness.

Background: The human gut microbiome contains millions of genes and many undetected bacteria species. Recovering bacterial genomes from large complex metagenomes remains highly challenging, and current binning methods show insufficient recall rates.

Objective: This study was performed to put forward a new metagenome binning method with promising recall rate and accuracy.

Delivering Molecular Beacons via an Electroporation-Based Approach Enables Live-Cell Imaging of Single RNA Transcripts and Genomic Loci.

Molecular beacons (MBs) are synthetic oligonucleotide probes that are designed to fluoresce upon hybridization to complementary nucleic acid targets. In contrast to genetically encoded probes that can be readily introduced into cells via standard transfection procedures, using MBs to obtain reliable intracellular measurements entails a reliable delivery method that maximizes MB entry while minimizing cell damage. One promising approach is microporation, a microliter volume electroporation-based method that exhibits reduced harmful events as compared with traditional electroporation methods.

The applications of nanopores in studies of proteins.

Nanopores are a label-free platform with the ability to detect subtle changes in the activities of individual biomolecules under physiological conditions. Here, we comprehensively review the technological development of nanopores, focusing on their applications in studying the physicochemical properties and dynamic conformations of peptides, individual proteins, protein-protein complexes and protein-DNA complexes. This is followed by a brief discussion of the potential challenges that need to be overcome before the technology can be widely accepted by the scientific community.

CRISPR/dual-FRET molecular beacon for sensitive live-cell imaging of non-repetitive genomic loci.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic imaging systems predominantly rely on fluorescent protein reporters, which lack the optical properties essential for sensitive dynamic imaging. Here, we modified the CRISPR single-guide RNA (sgRNA) to carry two distinct molecular beacons (MBs) that can undergo fluorescence resonance energy transfer (FRET) and demonstrated that the resulting system, CRISPR/dual-FRET MB, enables dynamic imaging of non-repetitive genomic loci with only three unique sgRNAs.

Live-Cell Imaging of Long Noncoding RNAs Using Molecular Beacons.

Long noncoding RNAs (lncRNAs) are a family of non-protein-coding RNA transcripts greater than 200 nucleotides in length that have been regarded as crucial modulators of gene expression in various biological and disease contexts, but mechanisms underlying such regulation still remains largely elusive. In addition to cell lysate-based approaches that have proven invaluable for studies of lncRNAs, live-imaging methods can add value by providing more in-depth information on lncRNA dynamics and localizations at the single-molecule level. Recently, we have developed a versatile imaging approach based on molecular beacons (MBs), which are a class of fluorogenic oligonucleotide-based probes with the capacity to convert RNA target hybridization into a measurable fluorescence signal.

Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms.

Chromatin conformation, localization, and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states, the requirement for cell fixation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context.

Single-Molecule Analysis of RNA Dynamics in Living Cells Using Molecular Beacons.

Over the past decade, emerging evidence has indicated that long intergenic noncoding RNAs (lincRNAs), a class of RNA transcripts greater than 200 nt in length, function as key regulators of gene expression in cellular physiology and pathogenesis. Greater understanding of lincRNA activities, particularly in the context of subcellular localization and dynamic regulation at the single-molecule level, is expected to provide in-depth understanding of molecular mechanisms that regulate cell behavior and disease evolution. We have recently developed a fluorescence-imaging approach to investigate RNA dynamics in living cells at the single-molecule level.

Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy.

During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (Gag) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly.

A CRISPR/molecular beacon hybrid system for live-cell genomic imaging.

The clustered regularly interspersed short palindromic repeat (CRISPR) gene-editing system has been repurposed for live-cell genomic imaging, but existing approaches rely on fluorescent protein reporters, making sensitive and continuous imaging difficult. Here, we present a fluorophore-based live-cell genomic imaging system that consists of a nuclease-deactivated mutant of the Cas9 protein (dCas9), a molecular beacon (MB), and an engineered single-guide RNA (sgRNA) harboring a unique MB target sequence (sgRNA-MTS), termed CRISPR/MB. Specifically, dCas9 and sgRNA-MTS are first co-expressed to target a specific locus in cells, followed by delivery of MBs that can then hybridize to MTS to illuminate the target locus.

Optimizing Molecular Beacons for Intracellular Analysis of RNA.

Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to confer greater biostability. We have recently developed a new MB architecture composed of 2'-O-methyl RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester stem (2Me/PS MB).

Quantifying Gene Expression in Living Cells with Ratiometric Bimolecular Beacons.

Molecular beacons (MBs), a class of oligonucleotide-based probes, have enabled researchers to study various RNA molecules in their native live-cell contexts. However, it is also increasingly recognized that, when delivered into cells, MBs have the tendency to be sequestered into the nucleus where they may generate false positive signals. In an attempt to overcome this issue, MBs have been synthesized with chemically modified oligonucleotide backbones to confer greater biostability.

Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane.

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear.

Engineering Novel Molecular Beacon Constructs to Study Intracellular RNA Dynamics and Localization.

With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss techniques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons (MBs), a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells.

Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation.

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation.

A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level.

Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering.

Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2'-O-methyl RNA molecular beacons.

Molecular Beacons (MBs) composed of 2'-O-methyl RNA (2Me) and phosphorothioate (PS) linkages throughout the backbone (2Me/PSFULL MBs) have enabled long-term imaging of RNA in living cells, but excess PS modification can induce nonspecific binding, causing false-positive signals. In this study, we evaluate the intracellular stability of MBs composed of 2Me with various PS modifications, and found that false-positive signals could be reduced to marginal levels when the MBs possess a fully PS-modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). Additionally, 2Me/PSLOOP MBs exhibited uncompromised hybridization kinetics, prolonged functionality and >88% detection accuracy for single RNA transcripts, and could do so without interfering with gene expression or cell growth.