Improving prime editing with an endogenous small RNA-binding protein.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Solution Structure of Poly(UG) RNA.

Poly(UG) or "pUG" RNAs are UG or GU dinucleotide repeat sequences which are highly abundant in eukaryotes. Post-transcriptional addition of pUGs to RNA 3' ends marks mRNAs as vectors for gene silencing in C. elegans. We previously determined the crystal structure of pUG RNA bound to the ligand N-methyl mesoporphyrin IX (NMM), but the structure of free pUG RNA is unknown. Here we report the solution structure of the free pUG RNA (GU)12, as determined by nuclear magnetic resonance spectroscopy and small and wide-angle x-ray scattering (NMR-SAXS-WAXS). The low complexity sequence and 4-fold symmetry of the structure result in overlapped NMR signals that complicate chemical shift assignment. We therefore utilized single site-specific deoxyribose modifications which did not perturb the structure and introduced well-resolved methylene signals that are easily identified in NMR spectra. The solution structure ensemble has a root mean squared deviation (RMSD) of 0.62 Å and is a compact, left-handed quadruplex with a Z-form backbone, or "pUG fold." Overall, the structure agrees with the crystal structure of (GU)12 bound to NMM, indicating the pUG fold is unaltered by docking of the NMM ligand. The solution structure reveals conformational details that could not be resolved by x-ray crystallography, which explain how the pUG fold can form within longer RNAs.

Interactions of arene ruthenium(II) complexes [η6-(C6H6)Ru(pprip)Cl]+ and [η6-(C6H6)Ru(H2iiP)Cl]+ with RNA triplex poly(U)•poly(A)*poly(U).

Two arene ruthenium(II) complexes [η6-(C6H6)Ru(pprip)Cl]PF6 (Ru1; pprip = 2-(3-phenyl-1H-pyrazol-4-yl)-imidazolo[4,5-f][1,10]phenanthroline) and [η6-(C6H6)Ru(H2iiP)Cl]PF6 (Ru2; H2iiP = 2-(indole-3-yl)-imidazolo[4,5-f][1,10]phenanthroline) have been synthesized and characterized in this work. Binding properties of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)*poly(U) were investigated by spectrophotometry and spectrofluorometry as well as viscosimetry. Analysis of spectroscopic titrations and viscosity measurements show that the two complexes bind with the triplex through intercalation, while the binding affinity for Ru2 toward the triplex is stronger than that for Ru1. Melting experiments indicate that the stabilizing effects of Ru1 and Ru2 toward the triplex differ from each other. Under the conditions used herein, Ru1 only stabilizes the Hoogsteen base-paired strand (third strand) without affecting stabilization of the Watson-Crick base-paired strand (the template duplex) of the triplex, while Ru2 stabilizes both the template duplex and the third strand. Although the two complexes prefer to stabilizing the third strand rather than the template duplex, the third-strand stabilization effect of Ru2 is stronger than that of Ru1. The obtained results of this work reveal that the planarity of the intercalative ligands plays an important role in the triplex stabilization by arene Ru(II) complexes.

Binding and stabilizating effect of RNA triplex poly(U)⋠poly(A)*poly(U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(dppx)]2.

Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. Spectrophotometric titrations and viscosity measurements suggested that enantiomers Λ-1 and Δ-1 bind with the triplex through intercalation, while the binding strengths of the two enantiomers toward the triplex differed only slightly from each other. Fluorescence titrations showed that although enantiomers Λ-1 and Δ-1 exhibited molecular "light switch" effects toward the triplex, the effect of Δ-1 was more marked. Furthermore, Furthermore, thermal denaturation showed that the two enantiomers have significantly different stabilizing effects on the triplex. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar to a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex. Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar as a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex.

Binding properties of a molecular "light switch" ruthenium(II) polypyridyl complex toward double- and triple-helical forms of RNA.

To further develop new luminescent probes for RNA, a new ruthenium(II) polypyridyl complex [Ru(dmb)2dppz-idzo]2+ (dmb = 4,4'-dimethyl-2,2'-bipyridine, dppz-idzo = dppz-imidazolone) has been synthesized and characterized in this study. Binding properties of [Ru(dmb)2dppz-idzo]2+ to RNA duplex poly(A) · poly(U) and triplex poly(U) · poly(A) ∗ poly(U) have been explored by spectroscopic techniques and viscometry experiments. The binding modes of [Ru(dmb)2dppz-idzo]2+ to RNA duplex and triplex are intercalation as revealed from spectral titrations and viscosity experiments, while the binding strength of this complex to duplex structure is significantly greater than that of triplex structure. Fluorescence titrations indicate that [Ru(dmb)2dppz-idzo]2+ can act as a molecular "light switch" for both duplex poly(A) · poly(U) and triplex poly(U) · poly(A) ∗ poly(U), while [Ru(dmb)2dppz-idzo]2+ is more sensitive to poly(A) · poly(U) compared to poly(U) · poly(A) ∗ poly(U) and poly(U). Therefore, this complex can distinguish between RNA duplex, triplex and poly(U), and can as luminescent probes for the three RNAs used in this study. In addition, thermal denaturation studies show that [Ru(dmb)2dppz-idzo]2+ is able to significantly increase the Stabilization of RNA duplex and triplex. The results obtained in this study may contribute to further understanding of the binding of Ru(II) complexes with different structural RNAs.

Insight into achirality and chirality effects in interactions of an racemic ruthenium(II) polypyridyl complex and its Δ- and Λ-enantiomers with an RNA triplex.

RNA triplexes have a variety of potential applications in molecular biology, diagnostics and therapeutics, while low stabilization of the third strand hinders their practical utilities under physiological conditions. In this regard, achieving the third-strand stabilization by binding small molecules is a promising strategy. Chirality is one of the basic properties of nature. To clarify achirality and chirality effects on the binding and stabilizing effects of RNA triplexes by small molecules, we report for the first time the RNA interactions of an racemic ruthenium(II) polypyridyl complex [Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) and its two enantiomers Δ/Λ-[Ru(bpy)2(11-CN-dppz)]2+ (Δ/Λ-Ru1) with an RNA triplex poly(U-A*U) (where "-" represents Watson-Crick base pairing, and "*" denotes Hoogsteen base pairing, respectively) in this work. Research shows that although rac-Ru1 and its two enantiomers Δ/Λ-Ru1 bind to the RNA triplex through the same mode of intercalation, the binding affinity for enantiomer Δ-Ru1 is much higher than that for rac-Ru1 and enantiomer Λ-Ru1. However, compared to enantiomer Λ-Ru1, the binding affinity for rac-Ru1 does not show much of an advantage, which is slightly greater than that for the former. Thermal denaturation measurements reveal both rac-Ru1 and Δ-Ru1 to have a preference for stabilizing the third strand rather than the template duplex of the RNA triplex, while Λ-Ru1 stabilizes the RNA triplex without significant selectivity. Besides, the third-strand stabilizing effects by rac-Ru1 and Δ-Ru1 are not markedly different from each other, but more marked than that by Λ-Ru1. This work shows that the binding properties of the racemic Ru(II) polypyridyl complex with the RNA triplex are not simply an average of its two enantiomers, indicating potentially complicated binding events.

A naked-eye colorimetric molecular "light switch" based on ruthenium(II) polypyridyl complex [Ru(phen)2ttbd]2+ as binder and stabilizer for RNA duplex and triplex.

Binding of [Ru(phen)2ttbd]2+ (phen = 1,10-phenanthroline, ttbd = 4-(6-propenylpyrido-[3,2-a]- phenzain-10-yl-benzene-1,2-diamine) to the RNA triplex poly(U-A*U) (herein "-" and "*" refer to the Watson-Crick and Hoogsteen binding, respectively) and the duplex poly(A-U) have been investigated by spectral technology and viscosity method. Analysis of spectral titrations and viscosity experiments as well as melting measurements suggest that [Ru(phen)2ttbd]2+ binds to the studied RNA triplex and duplex through intercalation, while its binding constant toward the triplex is greater than the duplex. Luminescent titrations indicate that [Ru(phen)2ttbd]2+ can act as a molecular "light switch" for the two RNAs and the switch effect can be detected by the naked-eye. Moreover, the "light switch" can be repeatedly cycled off and on by adjusting the pH of the solution, whereas color change in the case of the triplex is more significant compared with the duplex. To our knowledge, [Ru(phen)2ttbd]2+ is the first small molecule capable of serving as a pH-controlled reversible visual molecular "light switch" for both the RNA triplex poly(U-A*U) and duplex poly(A-U). Thermal denaturation experiments suggest that [Ru(phen)2ttbd]2+ can obviously increase the triplex stabilization, while it stabilizing third-strand is more marked in comparison with the template duplex of the triplex, indicating this complex preferentially binds to third-strand. The obtained results may be useful for understanding the binding of Ru(II) polypyridyl complexes to RNAs.

Interaction of arene ruthenium(II) complexes [(η6-C6H6)Ru(L)Cl]PF6 (L = o-fpip and p-fpip) with the RNA triplex poly(U)*poly(A)•poly(U).

To comprehend the binding properties of η6-arene Ru(II) complexes with poly(U)*poly(A)•poly(U) triplex, two arene Ru(II) complexes with different fluorine substituent positions, [(η6-C6H6)Ru(o-fpip)Cl]PF6 (Ru1,η6-C6H6 = benzene ring, o-fpip = 2-(2'­fluorine) imidazo [4,5-f] Biver et al. (2008), Gupta et al. (2012) [1, 10] phenanthroline) and [(η6-C6H6)Ru(p-fpip)Cl]PF6 (Ru2,η6-C6H6 = benzene ring, o-fpip = 2-(4'­fluorine) imidazo [4,5-f] Biver et al. (2008), Gupta et al. (2012) [1, 10] phenanthroline), have been synthesized and characterized in this study. The binding of Ru1 and Ru2 with poly(U)*poly(A)•poly(U) triplex has been investigated by viscosity measurement and spectroscopic methods. Analysis of UV-Vis absorption spectral titrations suggests that Ru1 and Ru2 bind to the triplex through an intercalative mode, but the binding affinity of Ru2 is slightly higher than that of Ru1, which is also verified by viscosity and EB (ethidium bromide) competition measurements. Furthermore, the thermal denaturation experiment shows that Ru1 and Ru2 increase the third-strand stabilization to a similar extent. Interestingly, the two complexes have essentially no effect on the stabilization of the template duplex. Considering the structure of Ru1 and Ru2, conceivably besides the intercalation of ligand, the force stabilizing the triplex should also involve covalent binding and electrostatic interaction. The obtained results will contribute to our understanding of the interaction of arene Ru(II) complexes with the poly(U)*poly(A)•poly(U) triplex.

The distinct RNA-interaction modes of a small ZnF domain underlay TUT4(7) diverse action in miRNA regulation.

TUT4 and the closely related TUT7 are non-templated poly(U) polymerases required at different stages of development, and their mis-regulation or mutation has been linked to important cancer pathologies. While TUT4(7) interaction with its pre-miRNA targets has been characterized in detail, the molecular bases of the broader target recognition process are unclear. Here, we examine RNA binding by the ZnF domains of the protein. We show that TUT4(7) ZnF2 contains two distinct RNA binding surfaces that are used in the interaction with different RNA nucleobases in different targets, i.e that this small domain encodes diversity in TUT4(7) selectivity and molecular function. Interestingly and unlike other well-characterized CCHC ZnFs, ZnF2 is not physically coupled to the flanking ZnF3 and acts independently in miRNA recognition, while the remaining CCHC ZnF of TUT4(7), ZnF1, has lost its intrinsic RNA binding capability. Together, our data suggest that the ZnFs of TUT4(7) are independent units for RNA and, possibly, protein-protein interactions that underlay the protein's functional flexibility and are likely to play an important role in building its interaction network.

Effects of sequence motifs in the yeast 3' untranslated region determined from massively parallel assays of random sequences.

BACKGROUND: The 3' untranslated region (UTR) plays critical roles in determining the level of gene expression through effects on activities such as mRNA stability and translation. Functional elements within this region have largely been identified through analyses of native genes, which contain multiple co-evolved sequence features. RESULTS: To explore the effects of 3' UTR sequence elements outside of native sequence contexts, we analyze hundreds of thousands of random 50-mers inserted into the 3' UTR of a reporter gene in the yeast Saccharomyces cerevisiae. We determine relative protein expression levels from the fitness of transformants in a growth selection. We find that the consensus 3' UTR efficiency element significantly boosts expression, independent of sequence context; on the other hand, the consensus positioning element has only a small effect on expression. Some sequence motifs that are binding sites for Puf proteins substantially increase expression in the library, despite these proteins generally being associated with post-transcriptional downregulation of native mRNAs. Our measurements also allow a systematic examination of the effects of point mutations within efficiency element motifs across diverse sequence backgrounds. These mutational scans reveal the relative in vivo importance of individual bases in the efficiency element, which likely reflects their roles in binding the Hrp1 protein involved in cleavage and polyadenylation. CONCLUSIONS: The regulatory effects of some 3' UTR sequence features, like the efficiency element, are consistent regardless of sequence context. In contrast, the consequences of other 3' UTR features appear to be strongly dependent on their evolved context within native genes.

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

HCV poly U/UC sequence-induced inflammation leads to metabolic disorders in vulvar lichen sclerosis.

Vulvar lichen sclerosis (VLS) is a dermatologic disorder that affects women worldwide. Women with VLS have white, atrophic papules on the vulva. They suffer from life-long intense pruritus. Corticosteroids are the first-line of treatments and the most effective medicines for VLS. Although VLS has been speculated as an autoimmune disease for a long time, its pathogenesis and the molecular mechanism is largely unknown. We performed a comprehensive multi-omics analysis of paired samples from VLS patients as well as healthy donors. From the RNA-seq analysis, we found that VLS is correlated to abnormal antivirus response because of the presence of Hepatitis C Virus poly U/UC sequences. Lipidomic and metabolomic analysis revealed that inflammation-induced metabolic disorders of fatty acids and glutathione were likely the reasons for pruritus, atrophy, and pigment loss in the vulva. Thus, the present study provides an initial interpretation of the pathogenesis and molecular mechanism of VLS and suggests that metabolic disorders that affect the vulva may serve as therapeutic targets for VLS.

Arginine multivalency stabilizes protein/RNA condensates.

Biomolecular condensates assembled through liquid-liquid phase separation (LLPS) of proteins and RNAs are currently recognized to play an important role in cellular organization. Their assembly depends on the formation of a network of transient, multivalent interactions between flexible scaffold biomolecules. Understanding how protein and RNA sequences determine these interactions and ultimately regulate the phase separation is an open key challenge. Recent in vitro studies have revealed that arginine and lysine residues, which are enriched in most cellular condensates, have markedly distinct propensities to drive the LLPS of protein/RNA mixtures. Here, we employ explicit-solvent atomistic molecular dynamics simulations to shed light on the microscopic origin of this difference by investigating mixtures of polyU oligonucleotides with either polyR/polyK peptides. In agreement with experiments, our simulations indicate that arginine has a higher affinity for polyU than lysine both in highly diluted conditions and in concentrated solutions with a biomolecular density comparable to cellular condensate. The analysis of intermolecular contacts suggests that this differential behavior is due to the propensity of arginine side chains to simultaneously form a higher number of specific interactions with oligonucleotides, including hydrogen bonds and stacking interactions. Our results provide a molecular description of how the multivalency of the guanidinium group enables the coordination of multiple RNA groups by a single arginine residue, thus ultimately stabilizing protein/RNA condensates.

RNA Sequence and Structure Determinants of Pol III Transcriptional Termination in Human Cells.

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that multiple uracils at the end of RNA transcripts are required for Pol III termination, the effects of upstream RNA secondary structure in the nascent transcript on transcriptional termination is still unclear. To address this, we developed an in cellulo Pol III transcription termination assay using the recently developed Tornado-Corn RNA aptamer system to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. For transcription from Pol III type 3 promoters, we found that only poly-U tracts longer than the average length found in the human genome efficiently terminate Pol III transcription without RNA secondary structure elements. We observed that RNA secondary structure elements placed in proximity to shorter poly-U tracts induced termination, and RNA secondary structure by itself was not sufficient to induce termination. For Pol III type 2 promoters, we found that the shorter poly-U tract lengths of 4 uracils were sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

Interaction of double-stranded polynucleotide poly(A:U) with graphene/graphene oxide.

Hybrids formed by DNA/RNA and graphene family nanomaterials are considered as potentially useful multifunctional agents in biosensing and nanomedicine. In this work, we study the noncovalent interaction between double-stranded (ds) RNA, polyadenylic:polyuridylic acids (poly(A:U)) and graphene oxide/graphene (GO/Gr) using UV absorption spectroscopy and molecular dynamics (MD) simulations. RNA melting showed that relatively long ds-RNA is adsorbed onto GO (at an ionic strength of [Formula: see text]) at that a large fraction of RNA maintains the duplex structure. It was revealed that this fraction decreases over long time (during a few days), indicating a slow adsorption process of the long polymer. MD simulations showed that the adsorption of duplex (rA)[Formula: see text]: (rU)[Formula: see text] or (rA)[Formula: see text]: (rU)[Formula: see text] on graphene starts with the interaction between [Formula: see text]-systems of graphene and base pairs located at a duplex tail. In contrast to relatively long duplex (rA)[Formula: see text]: (rU)[Formula: see text] which keeps parallel arrangement along the graphene surface, the shorter one ((rA)[Formula: see text]: (rU)[Formula: see text]) always adopts a perpendicular orientation relative to graphene even in case of the initial parallel orientation. It was found out that (rA)[Formula: see text]: (rU)[Formula: see text] forms the stable hybrid with graphene keeping essential fraction of the duplex, while (rA)[Formula: see text]: (rU)[Formula: see text] demonstrates the duplex unzipping into two single strands with time. The interaction energies between adenine/uracil stacked with graphene as well between nucleotides in water environment were determined.

Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.

Phase II Trial of Adjuvant Dendritic Cell Vaccine in Combination with Celecoxib, Interferon-α, and Rintatolimod in Patients Undergoing Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy for Peritoneal Metastases.

BACKGROUND: Peritoneal metastases portend poor prognosis in the setting of standard chemotherapy. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) improves outcomes, but relapse is common. We report a phase II trial evaluating the safety and efficacy of adjuvant αDC1 vaccination with chemokine modulation (CKM) after CRS/HIPEC. METHODS: Patients undergoing CRS/HIPEC for appendiceal cancer, colorectal cancer, or peritoneal mesothelioma were enrolled. In addition to standard adjuvant chemotherapy, patients received intranodal and intradermal injections of autologous tumor-loaded αDC1 vaccine. After each vaccine booster, patients received CKM over 4 days, consisting of celecoxib, interferon (IFN)-α, and rintatolimod. RESULTS: Forty-six patients underwent CRS/HIPEC followed by αDC1 treatment, including 24 appendiceal primaries, 20 colorectal, and 2 mesotheliomas. DC maturation was successful, with 97% expressing HLA-DR and CD86. Tumor cell recovery from peritoneal tumors was challenging, resulting in only 17% of patients receiving the target dose of αDC1. The αDC1 and CKM regimen was well tolerated. CKM successfully modulated serum inflammatory cytokine and chemokine levels. Median progression-free survival (PFS) for appendiceal primaries was 50.4, 34.2, and 8.9 months for grade 1, 2, and 3 tumors, respectively, while median PFS for colorectal cancer was 20.5 and 8.9 months for moderately and poorly differentiated tumors, respectively. CONCLUSIONS: Adjuvant autologous tumor antigen-loaded αDC1 vaccine and CKM is well tolerated. The mucinous nature of peritoneal metastases limits the feasibility of obtaining adequate autologous tumor cells. The improvement in median PFS did not meet our predefined thresholds, leading us to conclude that αDC1 vaccination is not appropriate for patients undergoing CRS/HIPEC for peritoneal metastases.

Effect of disease duration in a randomized Phase III trial of rintatolimod, an immune modulator for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

BACKGROUND: Rintatolimod is a selective TLR3 agonist, which has demonstrated clinical activity for ME/CFS in Phase II and Phase III double-blind, placebo-controlled, randomized, multi-site clinical trials. METHODS AND FINDINGS: A hypothesis-based post-hoc analysis of the Intent to Treat (ITT) population diagnosed with ME/CFS from 12 independent clinical sites of a Phase III trial was performed to evaluate the effect of rintatolimod therapy based on disease duration. The clinical activity of rintatolimod was evaluated by exercise treadmill tolerance (ETT) using a modified Bruce protocol. The ITT population (n = 208) was divided into two subsets of symptom duration. Patients with symptom duration of 2-8 years were identified as the Target Subset (n = 75); the remainder (<2 year plus >8 year) were identified as the Non-Target Subset (n = 133). Placebo-adjusted percentage improvements in exercise duration and the vertical rise for the Target Subset (n = 75) were more than twice that of the ITT population. The Non-Target Subset (n = 133) failed to show any clinically significant ETT response to rintatolimod when compared to placebo. Within the Target Subset, 51.2% of rintatolimod-treated patients improved their exercise duration by ≥25% (p = 0.003) despite reduced statistical power from division of the original ITT population into two subsets. CONCLUSION/SIGNIFICANCE: Analysis of ETT from a Phase III trial has identified within the ITT population, a subset of ME/CFS patients with ≥2 fold increased exercise response to rintatolimod. Substantial improvement in physical performance was seen for the majority (51.2%) of these severely debilitated patients who improved exercise duration by ≥25%. This magnitude of exercise improvement was associated with clinically significant enhancements in quality of life. The data indicate that ME/CFS patients have a relatively short disease duration window (<8 years) to expect a significant response to rintatolimod under the dosing conditions utilized in this Phase III clinical trial. These results may have direct relevance to the cognitive impairment and fatigue being experienced by patients clinically recovered from COVID-19 and free of detectable SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00215800.

A phenolic hydroxyl in the ortho- and meta-positions on the main ligands effect on the interactions of [Ru(phen)2(o-HPIP)]2+ and [Ru(phen)2(m-HPIP)]2+ with the poly(U)·poly(A)*poly(U) triplex.

The association of two ruthenium(II) complexes [Ru(phen)2(o-HPIP)]2+ (Ru1; phen = 1,10-phenanthroline, o-HPIP = 2-(2-hydroxyphenyl)-imidazo[4,5-f][1,10] phenanthroline) and [Ru(phen)2(m-HPIP)]2+ (Ru2; m-HPIP = 2-(3-hydroxyphenyl)-imidazo[4,5-f][1,10]phenan- throline) with the RNA poly(U)·poly(A)⁎poly(U) triplex has been investigated by spectrophotometric titrations and melting experiments in this work. All experimental data reveal an intercalative triplex-binding mode of the two complexes, whereas the binding constant for Ru1 is significantly higher than that for Ru2. Circular dichroism spectroscopic investigations show that the two complexes could bind to the chiral environment of the triplex, but the triplex perturbation effects induced by Ru1 are more marked. Thermal denaturation experiments demonstrate that both Ru1 and Ru2 display a large binding preference and stabilizing effect for the third strand over the Watson-Crick base-paired duplex of the triplex. However, the third-strand stabilizing effect of Ru1 is much more effective than that of Ru2. The obtained results suggest that positions of the phenolic group on the main ligands have significant effect on the binding of the two complexes with poly(U)·poly(A)⁎poly(U) triplex.

poly(UG)-tailed RNAs in genome protection and epigenetic inheritance.

Mobile genetic elements threaten genome integrity in all organisms. RDE-3 (also known as MUT-2) is a ribonucleotidyltransferase that is required for transposon silencing and RNA interference in Caenorhabditis elegans1-4. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (designated poly(UG) or pUG tails)5. Here we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to targets of RNA interference, as well as to transposon RNAs. RNA fragments attached to pUG tails with more than 16 perfectly alternating 3' U and G nucleotides become gene-silencing agents. pUG tails promote gene silencing by recruiting RNA-dependent RNA polymerases, which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed pUG RNA biogenesis underlie double-stranded-RNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA-siRNA silencing loop enables parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.