Forced intercalation-induced light-up peptides as fluorogenic indicators for the HIV-1 TAR RNA-ligand assay.

Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9-mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto a Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λem = 541 nm, Φfree = 0.0057, and Φbound = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP maintains strong binding affinity (Kd = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between "competitive" and "noncompetitive" inhibitors when used in the fluorescence indicator displacement (FID) assay. The FID assay under stringent screening conditions is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.

Design of synthetic peptide-based fluorescence probes for turn-on detection of hyaluronan.

Herein, we designed and examined a series of fluorescent peptide-based probes for turn-on detection of hyaluronan (HA), a member of the glycosaminoglycan family. We utilized two kinds of synthetic HA-binding peptides as the binding unit for HA, and each peptide was coupled with three kinds of environment-sensitive fluorophores as the signaling unit. From the examination of the peptides, fluorophores, and the position and number of fluorophore modification, we found that X7 peptide (RYPISRPRKR) labelled with an aggregation-induced emission (AIE) fluorogen, tetraphenylethene (TPE), at the N-terminal (named TPE-X7) did function as a light-up probe for HA. The response of TPE-X7 was highly selective to higher molecular weight HA in comparison with lower ones, having the possible potential for the analysis of HA size. TPE-X7 was also applicable to the quantification of HA in synovial fluids.

Synthetic DNA binders for fluorescent sensing of thymine glycol-containing DNA duplexes and inhibition of endonuclease activity.

Dimethyllumazine (DML)-thiazole orange (TO) conjugates were developed for fluorescence sensing of thymine glycol (Tg)-containing DNAs based on the selective recognition of the A nucleobase opposite the Tg residue. Additionally, this conjugate has demonstrated an inhibitory activity towards endonuclease III, a DNA repair enzyme, through its competitive binding to Tg-containing DNAs.

Kinetic analysis of highly effective triplex formation between a small molecule-peptide nucleic acid conjugate probe and the influenza A virus RNA promoter region at neutral pH.

In order to overcome the pH limitations of triplex-forming peptide nucleic acid (PNA) in binding to double-stranded RNA (dsRNA), we have recently proposed a new design of triplex-forming PNA-based fluorogenic probes that work at neutral pH for sensing the panhandle structure of the influenza A virus (IAV) RNA promoter region. Our strategy is based on the conjugation of a small molecule (DPQ) capable of selectively binding to the internal loop structure with the triplex-forming forced intercalation of thiazole orange (tFIT) probe with natural PNA nucleobases. In this work, the triplex formation of tFIT-DPQ conjugate probes with IAV target RNA at neutral pH was examined by means of a stopped-flow technique UV melting and fluorescence titration experiments. The obtained results revealed that (i) the conjugation strategy is responsible for the observed strong binding affinity due to a very fast association rate constant and a slow dissociation rate constant; (ii) the binding follows a pattern of the DPQ unit binding first to the internal loop region, followed by the tFIT unit binding to the complementary dsRNA region. Our results emphasize the importance of both the tFIT and the DPQ components of the conjugate probe design and revealed an association mechanism for the tFIT-DPQ probe-dsRNA triplex formation towards the IAV RNA at neutral pH.

Cationic Oligopeptides with Amino Groups as Synthetic Nucleolar Localization Signals for the Rational Design of Nucleolus-Staining Probes.

Cationic oligopeptides with amino groups were found to function as synthetic nucleolar localization signals for directing various fluorophores to the nucleolus with high selectivity in the cells with a view toward the development of nucleolus-staining probes.

Inhibition of SARS-CoV-2 nucleocapsid protein-RNA interaction by guanosine oligomeric RNA.

The interaction of the ß-coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein with genomic RNA is initiated by specific RNA regions and subsequently induces the formation of a continuous polymer with characteristic structural units for viral formation. We hypothesized that oligomeric RNAs, whose sequences are absent in the 29.9-kb genome sequence of SARS-CoV-2, might affect RNA-N protein interactions. We identified two such hexameric RNAs, In-1 (CCGGCG) and G6 (GGGGGG), and investigated their effects on the small filamentous/droplet-like structures (< a few µm) of N protein-genomic RNA formed by liquid-liquid phase separation. The small N protein structures were sequence-specifically enhanced by In-1, whereas G6 caused them to coalesce into large droplets. Moreover, we found that a guanosine 12-mer (G12, GGGGGGGGGGGG) expelled preexisting genomic RNA from the small N protein structures. The presence of G12 with the genomic RNA suppressed the formation of the small N protein structures, and alternatively apparently altered phase separation to induce the formation of large droplets with unclear phase boundaries. We showed that the N-terminal RNA-binding domain is required for the stability of the small N protein structures. Our results suggest that G12 may be a strong inhibitor of the RNA-N protein interaction.

Self-Assembly and Disassembly of Membrane Curvature-Sensing Peptide-Based Deep-Red Fluorescent Probe for Highly Sensitive Sensing of Exosomes.

With increasing knowledge of the diverse roles of exosomes in biological processes, much attention has been paid to the development of analytical methods for exosome analysis. Here, we developed a new class of amphipathic helical (AH) peptide-based fluorescent probes for highly sensitive detection of exosomes in a mix and read manner. Membrane curvature-sensing AH peptide (ApoC) was coupled with lipophilic tail (C12)-carrying thiazole red (TR) for construction of a self-assembly/disassembly based fluorescence "off-on" sensing system for target exosomes. ApoC-TRC12 has extremely weak emission due to the formation of the aggregates, whereas it becomes emissive in response to the target exosomes through the binding-induced disassembly of ApoC-TRC12. We demonstrated that the C12 unit attached to the TR unit had a favorable effect on both fluorescence response (signal-to-background: S/B) and binding affinity. ApoC-TRC12 was applicable to rapid and simple detection of exosomes with high detection sensitivity (limit of detection ≈ 103 particles/µL).

Classical thiazole orange and its regioisomer as fluorogenic probes for nucleolar RNA imaging in living cells.

In contrast to well-established DNA-selective dyes for live cell imaging, RNA-selective dyes have been less developed owing to the challenges of making small molecules have RNA selectivity over DNA. Two kinds of dyes are now commercially available for nucleolar RNA imaging in cells, but these two dyes do not apply to living cells and have limited use in fixed and permeabilized cells. Herein, we report on thiazole orange (TO), a well-known nucleic acid stain, as a promising fluorogenic dye for nucleolar RNA imaging in living cells. TO shows clear response selectivity for RNA over DNA with a significant light-up property upon binding to RNA (λem = 532 nm, I/I0 = 580-fold, and Φbound/Φfree = 380) and is even applicable to wash-free imaging of living cells. More interestingly, 2TO, a regioisomer of TO in which the benzothiazole unit is connected to position 2 in the quinoline ring, performs much better (λem = 532 nm, I/I0 = 430-fold, Φbound/Φfree = 1200), having superior selectivity for RNA in both solution and living cells. The comparison with TO derivatives carrying different substituents at N1 of the quinoline ring reveals that the slight change in the TO framework significantly affects RNA selectivity, photostability and membrane permeability.

Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells.

Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.

Enzyme sensor for simultaneous determination of cholesterol, triglyceride and HDL cholesterol in serum.

A three-electrode lipid biosensor that simultaneously measures the total cholesterol (CHO), triglyceride (TG), and HDL cholesterol (HDL-C) in standard serum has been developed. The lipid biosensor is designed for clinical use where production cost, low sample requirement, portability, stability, and speed are high priorities. The device design filters out blood cells and lipoproteins from the serum, where the target molecules are catalyzed by enzymes encapsulated in carboxymethyl cellulose (CMC) to produce electron mediators, potassium ferrocyanide. These electron mediators were subsequently detected by amperometric determination. The sensor exhibit high selectivity towards the targets and can measure the target lipids in 4 min with 10 µL of serum. Lastly, the device can be stored up to 18 months with a minimal decrease in catalytic efficiency.

Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule.

We have developed a new class of triplex-forming peptide nucleic acid (PNA)-based fluorogenic probes for sensing of the panhandle structure of the influenza A virus (IAV) RNA promoter region. Here, a small molecule (DPQ) capable of selectively binding to the internal loop structure was conjugated with triplex-forming forced intercalation of the thiazole orange (tFIT) probe with natural PNA nucleobases. The resulting conjugate, tFIT-DPQ, showed a significant light-up response (83-fold) upon strong (Kd = 107 nM) and structure-selective binding to the IAV RNA promoter region under physiological conditions (pH 7.0, 100 mM NaCl). We demonstrated the conjugation of these two units through the suitable spacer was key to show useful binding and fluorogenic signaling functions. tFIT-DPQ facilitated the sensitive and selective detection of IAV RNA based on its binding to the promoter region. Furthermore, we found that tFIT-DPQ could work as a sensitive indicator for screening of test compounds targeting the IAV RNA promoter region in the fluorescence indicator displacement assay.

Spectroscopic, thermodynamic and kinetic analysis of selective triplex formation by peptide nucleic acid with double-stranded RNA over its DNA counterpart.

Unlike conventional triplex-forming oligonucleotide (TFO), triplex-forming peptide nucleic acid (PNA) can tightly bind with double-stranded RNA (dsRNA) than double-stranded DNA (dsDNA). Here, we performed spectroscopic, thermodynamic and kinetic experiments for triplex formation by PNA to examine different binding behaviors between PNA - dsRNA and PNA - dsDNA triplexes. We found 9-mer PNA (cytosine content of 66%) formed the thermally stable triplex with dsRNA compared to dsDNA over a wide range of pH (5.5-8.0), salt concentration (50-500 mM NaCl). Both the calorimetric binding constant and the association rate constant for dsRNA were larger than those for dsDNA, indicating the favorable association process for the PNA - dsRNA triplex formation. Comparison with the DNA/RNA heteroduplexes revealed that the DNA strand was detrimental to the triplex stability for PNA, a contrasting result for conventional TFO. The keys underlying the difference in the triplex formation of PNA with different duplexes appear to be the conformational adoptability and the geometric compatibility of PNA to fit the deep, narrow major groove of dsRNA and the helical rigidity difference of the duplexes. Our results emphasize the importance of both the sugar puckering of the duplex and the appropriate conformational flexibility of PNA for the triplex formation.

Deep-red fluorogenic cyanine dyes carrying an amino group-terminated side chain for improved RNA detection and nucleolar RNA imaging.

The introduction of an amino-group-terminated side chain into deep-red emissive benzo[c,d]indole-quinoline monomethine cyanine dye has led to the improved detection of RNAs as well as the imaging of nucleolar RNAs in cells.

Small molecule-PNA oligomer conjugates for rRNA A-site at neutral pH for FID assays.

A triplex-forming PNA oligomer conjugated with a naphthyridine derivative (ATMND-C2-NH2) showed high selectivity and strong binding for the bacterial rRNA A-site at pH 7.0 (Kd = 190 ± 72 nM), which was accompanied by fluorogenic signaling that allowed the potential use of this conjugate probe in fluorescent indicator displacement assays.

Conjugating pyrene onto PNA-based fluorescent probes for improved detection selectivity toward double-stranded siRNA.

We report on the design of new siRNA-binding fluorescent probes with the improved detection selectivity toward intact double-stranded siRNAs over single-stranded forms by the conjugation of pyrene unit into thiazole orange base surrogate-carrying peptide nucleic acid (PNA) that can simultaneously recognize the 3'-overhang and double-stranded sequences of target siRNAs.

Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing.

With increasing knowledge about the diverse roles of exosomes in the biological process, much attention has been paid to develop analytical methods for detection and quantification of exosomes. Immunoassays based on the recognition of exosomal protein markers by antibodies were widely used. However, considering that exosomal protein composition varies with the cell type, the protein markers should be carefully selected for a sensitive and selective analysis of target exosomes. Herein, we developed a new class of exosome-binding fluorogenic probes based on membrane curvature (MC) sensing of amphipathic helical (AH) peptides for exosome analysis without the need to use protein markers on the exosomal membranes. The C-terminal region of apolipoprotein A-I labeled with Nile red (ApoC-NR) exhibited a significant fluorescence enhancement upon selective binding to the highly curved membranes of synthetic vesicles. Circular dichroism (CD) measurements involving 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-2-dioleoyl-sn-glycerol (DOG) vesicles suggested that ApoC-NR recognizes the lipid packing defects in the surface of highly curved membranes via the hydrophobic insertion of the α-helix structure of the ApoC unit. ApoC-NR exhibited a stronger binding affinity for exosome-sized vesicles and a higher MC selectivity compared to all other previously reported peptide probes. ApoC-NR can be used in a simple and rapid "mix and read" analysis of various kinds of exosomes derived from different cell types (limit of detection: -105 particles/µL) without being influenced by the variation in the expression of the surface proteins of the exosomes, which stands in sharp contrast to immunoassays.

Deep-Red Light-up Signaling of Benzo[c,d]indole-Quinoline Monomethine Cyanine for Imaging of Nucleolar RNA in Living Cells and for Sequence-Selective RNA Analysis.

RNA-binding small probes with deep-red emission are promising for RNA analysis in biological media without suffering from background fluorescence. Here benzo[c,d]indole-quinoline (BIQ), an asymmetric monomethine cyanine analogue, was newly developed as a novel RNA-selective probe with light-up signaling ability in the deep-red spectral range. BIQ features a significant light-up response (105-fold) with an emission maximum at 657 nm as well as improved photostability over the commercially available RNA-selective probe, SYTO RNA select. BIQ was successfully applied to the fluorescence imaging of nucleolar RNAs in living cells with negligible cytotoxicity. Furthermore, we found the useful ability of BIQ as a base surrogate integrated in peptide nucleic acid (PNA) oligonucleotides for RNA sequence analysis. BIQ base surrogate functioned as a deep-red light-up base surrogate in forced intercalation (FIT) and triplex-forming FIT (tFIT) systems for the sequence-selective detection of single-stranded and double-stranded RNAs, respectively.

Strong Binding and Off-On Signaling Functions of Deep-Red Fluorescent TO-PRO-3 for Influenza†A Virus RNA Promoter Region.

The RNA promoter region of the influenza A virus has recently attracted much attention as an RNA target for the development of anti-influenza drugs. However, there are very few reports on small RNA-binding ligands targeting this region. In this work, it is reported that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, exhibits strong and selective binding to the internal loop structure of the influenza A virus RNA promoter. This binding accompanies the remarkable light-up response of TO-PRO-3 in the deep-red spectral region. By virtue of these binding and fluorescence signaling functions, TO-PRO-3 can act as a useful indicator for the assessment of the binding capabilities of various test compounds for this RNA region, with a view toward the development of anti-influenza drug candidates.

Trimethine cyanine dyes as deep-red fluorescent indicators with high selectivity to the internal loop of the bacterial A-site RNA.

We report that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, functions as a deep-red fluorescent indicator for the internal loop structure of the bacterial (Escherichia coli) ribosomal decoding region of the aminoacyl-tRNA site (A-site), which enables the assessment of A-site binding capability of various test compounds including blue and even-green-emitting compounds.

Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation.

RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable. Here, we focused on Broccoli, a superior variant of Spinach, which is a well-known fluorescent RNA aptamer that binds to DFHBI-1T and emits green fluorescence. We experimentally characterized Broccoli and predicted that it forms a G-quadruplex-based DFHBI-1T recognition region sandwiched between two stems. Based on this, we designed a Broccoli-based RNA detection probe (BRD probe) composed of a sequence of destabilized Broccoli fused with complementary sequences against target RNA. The resulting probe with its target RNA formed a stable three-way junction, named the MT2 three-way junction, which contributed to efficient refolding of the Broccoli structure and allowed for programmable RNA detection with high signal-to-noise ratio and sensitivity. Interestingly, the MT2 three-way junction also could be applied to probe construction of a truncated form of Spinach (Baby Spinach). The BRD and Baby Spinach-based RNA detection probes (BSRD probe) exhibited up to 48- and 140-fold fluorescence enhancements in the presence of their target RNAs and detected small amounts of target RNA that were as low as 160 and 5 nM, respectively. Thus, we experimentally characterized the higher order structure of Broccoli and developed structure-switching aptamer probes for highly sensitive, programmable, RNA detection using an MT2 three-way junction.