Guanidinium-linked morpholino-siRNA chimera: synthesis, biophysical properties and activity.

This study introduces guanidinium morpholino RNA (GMO-RNA), an RNA analogue with enhanced cell permeability, nuclease resistance and RNA interference activity. Self-penetrating GMO-RNA chimeras leverage guanidinium-mediated endocytic uptake across multiple cell lines. Thermal studies validated duplex stability, and Eg5-targeting GMO-siRNA achieved effective gene silencing in cervical cancer cells, demonstrating therapeutic potential.

An Open-Source Normothermic Perfusion System Designed for Research Scientists.

The integration of ex vivo perfusion machines for human organs prior to transplantation has improved outcomes for recipients and increased organ availability while revealing novel avenues of investigation for translational medicine. However, these machines have limited availability for experimental modifications while presenting a significant upfront cost investment and prohibitive per-use cost to the research scientist. Furthermore, there is a significant need for improved pre-clinical models in biomedical research that allow for authentic interrogation of cellular processes in the multi-cellular organ setting.

Thermodynamic Destabilization and Structural Adaptability of 2'-Alkoxy/Fluoro--Methyl Pyrimidine Nucleotides in siRNA Enhance RNA Interference Activity.

Herein, we describe the synthesis of 2'-alkoxy/fluoro-mC phosphoramidites and their incorporation into siRNAs. These modifications, particularly at the central position of the siRNA duplex, caused significant thermal destabilization due to disrupted Watson-Crick hydrogen bonding. 2'-Alkoxy-mC conferred improved resistance toward 5'- and 3'-exonucleases compared to their 2'-fluoro-mC and mC counterparts.

Expression of IMPACT curtails metabolic plasticity and augments NK cell killing to abrogate metastatic growth.

Given the propensity of aggressive epithelial tumors to form hepatic metastases, we performed an in vivo cDNA screen using the mouse liver and KRASG12D/TP53R273H pancreatic cells to identify the RNA binding protein GCN1 as integral component of hepatic outgrowth. RNAi experiments reveal that GCN1 triggers the ISR to activate serine, folate, and methionine biosynthetic pathways together with amino acid transporters, which act in concert to facilitate acquisition of metabolites and to restore redox homeostasis. Alongside activation of the ISR, we found that GCN1 also functions in the nucleus where it interacts with HNRNPK to suppress the expression of MHC-I molecules, and natural killer (NK) ligands.

Wireless Uncaging of Boronates in Living Cells via Piezocatalytically Generated HO.

Piezoelectric materials offer wireless bond cleavage in remote areas using ultrasound or other mechanical pressure. Although this approach has enormous therapeutic application potential, the appropriate design of such materials is limited and yet to be explored. In this study, we demonstrate the use of BaTiO nanoparticles for piezocatalytic bond cleavage and the controlled release of organic boronates within live cells, facilitated by medical-grade ultrasound.

4'-O-methoxyethyl-2'-deoxy-uridine and cytidine ribonucleotides improve duplex stability, nuclease resistance, and elicit efficient knockdown of Bcl-2 expression.

In this study, we report the synthesis of 4'-O-methoxyethyl deoxyuridine and 4'-O-methoxyethyl deoxycytidine phosphoramidites and their incorporation into oligonucleotides. The 3'-exonuclease experiment indicated a significant increase in stability with the incorporation of 4'-O-MOE dU alone (T = 178 min) and in combination with phosphorothioate modification (T = 13 h) in dT-mer. Western blot studies demonstrated that 4'-O-MOE-dU and 4'-O-MOE-dC modifications were well-tolerated when placed at both overhang regions and multiple positions within the passenger strand of anti-Bcl2 siRNAs.

Enhanced G-Quadruplex Stability and Anticancer Activity of -Aryl Deoxyguanosine-Modified T40214 Oligonucleotides Targeting STAT3 in Prostate Cancer.

Signal transducer and activator of transcription 3 (Stat3) is a key molecular target in many cancers due to its role in tumor cell proliferation and survival. T40214, a G-quadruplex (G4) forming oligonucleotide, targets the Stat3 dimer and inhibits its DNA binding activity. In this study, we introduced -furfuryl and -cinnamyl deoxyguanosine modifications at the G-tetrad positions in T40214 to assess their structural and antitumor effects in prostate cancer cells.

Phosphoramidite Platform for the Automated Synthesis of Morpholino Antisense Oligonucleotides and Their Chimeras.

Phosphorodiamidate morpholino oligonucleotides (PMOs) are antisense oligonucleotides that modulate gene expression through a steric blocking mechanism. Despite their increasing use in antisense therapeutics, the synthesis of PMOs remains challenging, thus limiting their accessibility. As a result, a modular synthesis protocol for PMOs, compatible with DNA/RNA synthesizers, is highly anticipated to facilitate the development of next-generation PMO chimeras incorporating other oligonucleotide backbones with proven therapeutic efficacy.

4'--Cholesterol/Pyridyl-2'--Methyl Uridine-Functionalized siRNA Enhances Stability and Carrier-Free Gene Silencing.

Chemical modifications and targeted delivery through the conjugation of small molecules have transformed the potential of siRNA-based therapeutics. These advancements address key challenges, such as poor cellular uptake, low bioavailability, and limited metabolic stability, making siRNA delivery more efficient and clinically viable. Cholesterol-conjugated siRNA enables cellular uptake through lipoprotein pathways without transfection agents.

Chemical Insights into Oligonucleotide-Protein Binding for Therapeutic Applications.

Plasma protein binding is an important determinant in the clinical success of oligonucleotide-based drugs. Optimal protein binding of the oligonucleotide is critical to its tissue distribution and retention by preventing renal excretion. This property can be modulated through suitable chemical modifications depending on the oligonucleotide backbone to achieve a balanced pharmacokinetic profile and minimize off-target effects.

Optochemical control over mRNA translation by photocaged phosphorodiamidate morpholino oligonucleotides in vivo.

We developed an efficient, robust, and broadly applicable system for light-induced protein translation to control the production of proteins of interest and study their function. The method is based on the displacement of a single type of antisense morpholino from RNA by the uncaged guanidinium-linked morpholino (GMO)-phosphorodiamidate morpholino oligonucleotide (PMO) chimera upon UV irradiation. The GMO-PMO chimera designed here is cell-permeable and the GMO part can be produced employing a mercury-free approach compatible with the synthesis on solid support.

Site-Specific Chemical Modulation of a Flexible Azaproline Transporter to Enhance Epirubicin Accumulation in Drug-Resistant Human Glioblastoma Cells and Blood-Brain Barrier Penetration in Adult Zebrafish.

Cell-penetrating peptides (CPPs) have emerged as nonviral biological carriers for the delivery of macromolecular therapeutics into cells. Despite their lower immunogenicity and cellular toxicity, CPPs often lack target specificity and proteolytic stability. Various modified CPPs have been reported to improve such selective targeting and pharmacokinetic properties .

A Unified Phosphoramidite Platform for the Synthesis of Morpholino Oligonucleotides and Diverse Chimeric Backbones.

Phosphorodiamidate Morpholino Oligonucleotides (PMOs) have been well established in the milieu of FDA-approved oligonucleotide-based drugs in the past decade. Given their relevance in antisense therapeutics, a DNA/RNA synthesizer-compatible modular synthesis protocol of PMOs is long awaited to explore next-generation PMO chimeras with other therapeutically proven oligonucleotide backbones. Herein, we demonstrate a streamlined 5' → 3'phosphoramidite approach for the synthesis of PMOs using -butyl-protected 5'-morpholino phosphoramidites, which were synthesized from 5'-OH morpholino monomers derived from commercially available ribonucleosides.

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies.

2'--Alkyl- -Methyluridine Functionalized Passenger Strand Improves RNAi Activity by Modulating the Thermal Stability.

Herein, we have demonstrated that the siRNA activity could be enhanced by incorporating the guide strand in the RISC complex through thermodynamic asymmetry caused by mU-based destabilizing modifications. A nuclease stability study revealed that 2'-OMe-mU and 2'-OEt-mU modifications slightly improved the half-lives of siRNA strands in human serum. In the gene silencing assay, 2'-OMe-mU modification at the 3'-overhang and cleavage site of the passenger strand in anti-renilla and anti-Bcl-2 siRNA duplexes were well-tolerated and exhibited improved gene silencing activity.

Total Synthesis of Lipopeptide Bacilotetrin C: Discovery of Potent Anticancer Congeners Promoting Autophagy.

A convergent strategy for the first total synthesis of the lipopeptide bacilotetrin C has been developed. The key features of this synthesis include Crimmins acetate aldol, Steglich esterification, and macrolactamization. Twenty-nine variants of the natural product were prepared following a systematic structure-activity relationship study, where some of the designed analogues showed promising cytotoxic effects against multiple human carcinoma cell lines.

Benzofuran Iboga-Analogs Modulate Nociception and Inflammation in an Acute Mouse Pain Model.

Pain management following acute injury or post-operative procedures is highly necessary for proper recovery and quality of life. Opioids and non-steroidal anti-inflammatory drugs (NSAIDS) have been used for this purpose, but opioids cause addiction and withdrawal symptoms whereas NSAIDS have several systemic toxicities. Derivatives of the naturally occurring iboga alkaloids have previously shown promising behavior in anti-addiction of morphine by virtue of their interaction with opioid receptors.

4'--Acetamidomethyl-2'--methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs.

In this study, we designed the 4'--acetamidomethyl-2'--methoxyethyl (4'--ACM-2'--MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'--ACM-2'--MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs.

Synthesis and Biophysical Properties of Triazole-Incorporated PMOs (TzPMOs): A Convergent, Click Ligation Approach.

The synthesis of phosphorodiamidate morpholino oligonucleotides (PMOs) incorporating single or double triazole rings in the backbone has been achieved via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The synthetic approach implemented is fundamentally convergent, involving the ligation of a 5'-azide PMO fragment to a 3'-alkyne fragment both in solution and on solid support. To access the 3'-alkyne PMO fragment, we synthesized 3'--propargyl chlorophosphoramidate morpholino monomers for all four nucleobases.

Synthesis of Self Permeable Antisense PMO Using C5-Guanidino-Functionalized Pyrimidines at the 5'-End Enables Sox2 Downregulation in Triple Negative Breast Cancer Cells.

Delivery of macromolecular drugs inside cells has been a huge challenge in the field of oligonucleotide therapeutics for the past few decades. Earliest natural inspirations included the arginine rich stretch of cell permeable HIV-TAT peptide, which led to the design of several molecular transporters with varying numbers of rigid or flexible guanidinium units with different tethering groups. These transporters have been shown to efficiently deliver phosphorodiamidate morpholino oligonucleotides, which have a neutral backbone and cannot form lipoplexes.

N-Methyluridine and 2'-O-Alkyl/2'-Fluoro-N-methyluridine functionalized nucleic acids improve nuclease resistance while maintaining duplex geometry.

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N-methyluridine (2'-O-alkyl/2'-F-mU) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-mU modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT with 2'-O-alkyl-mU modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to mU, 2'-F, 2'-OMe modified oligonucleotides.

Outcome Predictors of Percutaneous Cholecystostomy As Definitive Versus Bridging Treatment for Acute Cholecystitis.

Introduction Percutaneous cholecystostomy (PC) is a treatment option for patients with acute cholecystitis (AC) who are too unwell, or too morbid for laparoscopic cholecystectomy (LC). Some patients have PC as a definitive treatment, whereas others have PC as a bridging treatment prior to LC. The aim of this study is to investigate patient characteristics and mortality among those who received PC as definitive treatment versus bridging treatment.

Morpholino oligonucleotide-mediated exon skipping for DMD treatment: Past insights, present challenges and future perspectives.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease primarily affecting boys causing loss of the dystrophin protein, ultimately leading to muscle wastage and death by cardiac or respiratory failure. The genetic mutation involved can be overcome with antisense oligonucleotides which bind to a pre-mRNA and results in reading frame restoration by exon skipping. Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense agents with a neutral backbone derived from RNA which can induce effective exon skipping.

Synthesis and Biophysical Studies of High-Affinity Morpholino Oligomers Containing G-Clamp Analogs.

Successful syntheses of chlorophosphoramidate morpholino monomers containing tricyclic cytosine analogs phenoxazine, G-clamp, and G-clamp were accomplished. These modified monomers were incorporated into 12-mer oligonucleotides using trityl-chemistry by an automated synthesizer. The resulting phosphorodiamidate morpholino oligomers, containing a single G-clamp, demonstrated notably higher affinity for complementary RNA and DNA compared to the unmodified oligomers under neutral and acidic conditions.

Hydroalcoholic root extracts of Houttuynia cordata (Thunb.) standardized by UPLC-Q-TOF-MS/MS promotes apoptosis in human hepatocarcinoma cell HepG2 via GSK-3β/β-catenin/PDL-1 axis.

Houttuynia cordata (Thunb.), an important medicinal plant of Northeast India, Korea, and China, is used to treat various ailments and for anticancer research. Knowing its traditional practices, we are interested in the mode-of-action of HCT on HepG2 to co-relate the traditional practice with modern drug therapeutics.