A Lucifer‐Based Environment‐Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs

Fluorescence‐based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1) a new microenvironment‐sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore ( 1,8‐naphthalimide) at the 5‐position of uracil, and 2) a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells—poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.     

Can Phosphate‐Branched RNA Persist under Physiological Conditions?

A 2′‐O‐methyl‐RNA oligonucleotide containing a single free 2′‐OH group flanking a branching phosphotriester linkage was prepared as a model for phosphate‐branched RNA by using an orthogonally protected dimeric phosphoramidite building block in solid‐phase synthesis. The strategy allows the synthesis of phosphate‐branched oligonucleotides, the three branches of which may be of any desired sequence. Hydrolytic reactions of the phosphotriester linkages in such oligonucleotides were studied at physiological pH in the presence (and absence) of various complementary oligonucleotides. The fully hybridized oligonucleotide model is an order of magnitude more stable than its single‐stranded counterpart, which, in turn, is an order of magnitude more stable than its trinucleoside phosphotriester core lacking any oligonucleotide arms. Furthermore, kinked structures obtained by hybridizing the phosphate‐branched oligonucleotide with partially complementary oligonucleotides are three to five times more stable than fully double‐stranded ones and only approximately three times less stable than the so‐called RNA X structure, which has been postulated to incorporate an RNA phosphotriester linkage. The results indicate that when the intrinsically unstable RNA phosphotriester linkage is embedded in an oligonucleotide of appropriate tertiary structure, its half‐life can be at least several hours.     

Novel (phenylethynyl)pyrene-LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets

We describe fluorescent oligonucleotide probes labeled with novel (phenylethynyl)pyrene dyes attached to locked nucleic acids. Furthermore, we prove the utility of these probes for the effective detection of single-nucleotide polymorphisms in natural nucleic acids. High-affinity hybridization of the probes and excellent fluorescence responses to single-base mismatches in DNA/RNA targets are demonstrated in model dual-probe and doubly labeled probe formats. This stimulated us to develop two diagnostic systems for the homogeneous detection of a drug-resistance-causing mutation in HIV-1 protease cDNA and RNA gene fragments. Target sequences were obtained by analysis of 200 clinical samples from patients currently receiving anti-HIV/AIDS combination therapy at the Russian Federal AIDS Center. Using these fluorescent oligonucleotides, we were able to detect the target mutation despite all the challenges of the natural targets, that is, the presence of additional mutations, neighboring sequence variation, and low target concentration, which typically reduce binding and effectiveness of sensing by fluorescent oligonucleotides.     

2'-O-Lysylaminohexyl oligonucleotides: modifications for antisense and siRNA

A novel type of oligonucleotide has been developed, characterized by the attachment of a lysyl moiety to a 2'-O-aminohexyl linker. A protected lysine building block was tethered to 2'-O-aminohexyluridine, and the product was converted into the corresponding phosphoramidite. Up to six modified nucleosides were incorporated in dodecamer DNA and RNA oligonucleotides using standard phosphoramidite chemistry. Each of the building blocks contributes one positive charge to the oligonucleotide instead of the negative charge of a wild-type nucleotide. Thermal denaturation profiles indicated a stabilizing effect of 2'-O-lysylaminohexyl chains that was more pronounced in RNA duplexes. Incubation of the oligonucleotides with 5'-exonuclease revealed an exceptionally high stability against enzymatic degradation. Incorporation of up to three modifications into functional antisense and siRNA oligonucleotides targeted at ICAM-1 showed that the gene-silencing activity was higher with an increasing number of lysylaminohexyl nucleotides. Compared with wild-type antisense or siRNA, compounds with three modifications led to equal or higher ICAM-1 downregulation.     

Evaluation of bacterial RNase P RNA as a drug target

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense-based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3'-CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA-CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3') complementary to the CCA binding site and its 3' flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3' and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3' with this region and occurs in a highly sequence-specific manner. A DNA version of Eco 3' was a less potent inhibitor. The potential of Eco 3' to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11-J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11-J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.     

Molecular Selection,Modification and Development of Therapeutic Oligonucleotide Aptamers

Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the detailed advantages and disadvantages of antibodies and aptamers in therapeutic applications and summarize recent progress in aptamer selection and modification approaches. We will present therapeutic oligonucleotide aptamers in preclinical studies for skeletal diseases and further discuss oligonucleotide aptamers in different stages of clinical evaluation for various disease therapies including macular degeneration, cancer, inflammation and coagulation to highlight the bright commercial future and potential challenges of therapeutic oligonucleotide aptamers.     

Recent Advances in Oligonucleotide Therapeutics in Oncology

Cancer is one of the leading causes of death worldwide. Conventional therapies, including surgery, radiation, and chemotherapy have achieved increased survival rates for many types of cancer over the past decades. However, cancer recurrence and/or metastasis to distant organs remain major challenges, resulting in a large, unmet clinical need. Oligonucleotide therapeutics, which include antisense oligonucleotides, small interfering RNAs, and aptamers, show promising clinical outcomes for disease indications such as Duchenne muscular dystrophy, familial amyloid neuropathies, and macular degeneration. While no approved oligonucleotide drug currently exists for any type of cancer, results obtained in preclinical studies and clinical trials are encouraging. Here, we provide an overview of recent developments in the field of oligonucleotide therapeutics in oncology, review current clinical trials, and discuss associated challenges.     

Phenol/Chloroform-Free TiO2-Based miRNA Extraction from Cell Lysate

While microRNAs are considered as excellent biomarkers of various diseases, there are still several remaining challenges regarding their isolation. In this study, we aimed to design a novel RNA isolation method that would help to overcome those challenges. Therefore, we present a novel phenol/chloroform-free, low-cost method for miRNA extraction. Within this method, RNA is extracted from cell lysate with an isopropanol/water/NaCl system, followed by solid-phase extraction using TiO₂ microspheres to effectively separate short RNAs from long RNA molecules. We also demonstrated the pH-dependent selectivity of TiO₂ microspheres towards different sizes of RNA. We were able to regulate the size range of extracted RNAs with simple adjustments in binding conditions used during the solid-phase extraction.     

Synthesis, properties, and applications of oligonucleotides containing an RNA dinucleotide phosphorothiolate linkage

RNA represents a prominent class of biomolecules. Present in all living systems, RNA plays many essential roles in gene expression, regulation, and development. Accordingly, many biological processes depend on the accurate enzymatic processing, modification, and cleavage of RNA. Understanding the catalytic mechanisms of these enzymes therefore represents an important goal in defining living systems at the molecular level. In this context, RNA molecules bearing 3'- or 5'-S-phosphorothiolate linkages comprise what are arguably among the most incisive mechanistic probes available. They have been instrumental in showing that RNA splicing systems are metalloenzymes and in mapping the ligands that reside within RNA active sites. The resulting models have in turn verified the functional relevance of crystal structures. In other cases, phosphorothiolates have offered an experimental strategy to circumvent the classic problem of kinetic ambiguity; mechanistic enzymologists have used this tool to assign precise roles to catalytic groups as general acids or bases. These insights into macromolecular function are enabled by the synthesis of nucleic acids bearing phosphorothiolate linkages and the unique chemical properties they impart. In this Account, we review the synthesis, properties, and applications of oligonucleotides and oligodeoxynucleotides containing an RNA dinucleotide phosphorothiolate linkage. Phosphorothioate linkages are structurally very similar to phosphorothiolate linkages, as reflected in the single letter of difference in nomenclature. Phosphorothioate substitutions, in which sulfur replaces one or both nonbridging oxygens within a phosphodiester linkage, are now widely available and are used routinely in numerous biochemical and medicinal applications. Indeed, synthetic phosphorothioate linkages can be introduced readily via a sulfurization step programmed into automated solid-phase oligonucleotide synthesizers. In contrast, phosphorothiolate oligonucleotides, in which sulfur replaces a specific 3'- or 5'-bridging oxygen, have presented a more difficult synthetic challenge, requiring chemical alterations to the attached sugar moiety. Here we begin by outlining the synthetic strategies used to access these phosphorothiolate RNA analogues. The Arbuzov reaction and phosphoramidite chemistry are often brought to bear in creating either 3'- or 5'-S-phosphorothiolate dinucleotides. We then summarize the responses of the phosphorothiolate derivatives to chemical and enzymatic cleavage agents, as well as mechanistic insights their use has engendered. They demonstrate particular utility as probes of metal-ion-dependent phosphotransesterification, general acid-base-catalyzed phosphotransesterification, and rate-limiting chemistry. The 3'- and 5'-S-phosphorothiolates have proven invaluable in elucidating the mechanisms of enzymatic and nonenzymatic phosphoryl transfer reactions. Considering that RNA cleavage represents a fundamental step in the maturation, degradation, and regulation of this important macromolecule, the significant synthetic challenges that remain offer rich research opportunities.     

Advanced Process Research and Development to Enhance Materials Recycling

Innovative, cost-effective technologies that have a positive life-cycle environmental impact and yield marketable products are needed to meet the challenges of the recycling industry. Two polymeric materials-recovery technologies that are being developed at Argonne National Laboratory in cooperation with industrial partners are described in this article.     

Stabilisation of RNA bulges by oligonucleotide complements containing an adenosine analogue

Incorporation of 2'-deoxy-2'-beta-(1-naphthylmethyl)tubercidin into an oligodeoxyribonucleotide mostly has little or a slightly negative effect on the T(m) values of complexes with DNA complements. With the same naphthylmethyl-substituted nucleoside at the 3'-end of a 2'-O-methyloligoribonucleotide, however, a stabilisation of 1-2 degrees C in the corresponding complexes with both DNA and RNA is observed. When the target sequence is an RNA fragment forming a two- or three-nucleotide bulge, complexes with (naphthylmethyl)tubercidin-modified oligodeoxyribonucleotides, as well as with the corresponding 2'-O-methyloligoribonucleotides, give stabilisations of 1-2 degrees C for the three-nucleotide bulge and of almost 4 degrees C for the two-nucleotide bulge. This stabilisation is specific to RNA, since the corresponding complexes with the DNA fragments do not display this effect. Thus, the (naphthylmethyl)tubercidin-containing oligonucleotides are the first reported oligonucleotide modifications that specifically stabilise bulged RNA.     

Nucleic Acid Aptamer‐Mediated Drug Delivery for Targeted Cancer Therapy

Aptamers are emerging as promising therapeutic agents and recognition elements. In particular, cell‐SELEX ( s ystematic e volution of l igands by ex ponential enrichment) allows in vitro selection of aptamers selective to whole cells without prior knowledge of the molecular signatures on the cell surface. The advantage of aptamers is their high affinitiy and binding specificity towards the target. This Minireview focuses on single‐stranded (ss) oligonucleotide (DNA or RNA)‐based aptamers as cancer therapeutics/theranostics. Specifically, aptamer–nanomaterial conjugates, aptamer–drug conjugates, targeted phototherapy and targeted biotherapy are covered in detail. In reviewing the literature, the potential of aptamers as delivery systems for therapeutic and imaging applications in cancer is clear, however, major challenges remain to be resolved, such as the poorly understood pharmacokinetics, toxicity and off‐target effects, before they can be fully exploited in a clinical setting.     

DNA as a Platform to Program Assemblies with Emerging Functions in Chemical Biology

The programmability of oligonucleotide recognition offers an attractive platform for directing the assembly of ligands that can interact cooperatively with a target or the assembly of reactive partner that can engage in chemical reactions. In both cases, an oligonucleotide directs the assembly, which yields a functional output. In terms of the controlled display of ligands, several examples have shown a significant increase in binding upon ligand assembly using small molecule fragments, peptides, glycans, and protein fragments. Combinatorial approaches to identifying the optimal pairing have also been reported. In terms of nucleic acid triggered reactions, several robust chemistries have been reported, leading to the unmasking of different fluorophores or bioactive molecules as well as the synthesis of a bioactive compound in response to DNA or even cellular RNA. Herein we present the historical context of this work and summarize recent developments in this area.     

Commercial challenges in developing biomaterials for medical device development

From initial conceptualization through to market adoption, the introduction of new technology into a commercial market is often fraught with challenges. In the field of biomaterials these challenges manifest themselves at every step of the commercialization process. As the understanding of the mechanobiologic relationship in tissue engineering has progressed, there is greater emphasis being placed on the engineering of specific properties into new biomaterials. During this early development, intellectual property is a critical factor in determining commercial feasibility as patent protection establishes the property lines around the technology and helps define future market segments. Once developed, a new biomaterial then enters the next series of challenges including commercial‐scale manufacturing and addressing regulatory concerns. Synthesis of the biomaterial must be economically and technically scalable to meet market demands and the new biomaterial must be designed to stand up to regulatory scrutiny with regards to safety and efficacy. However daunting, a robust understanding of the design elements and the commercial requirements will aid the researcher in overcoming the challenges of bringing a new biomaterial to the market. © 2018 Society of Chemical Industry     

Self-Amplifying RNA Approach for Protein Replacement Therapy

Messenger RNA (mRNA) technology has already been successfully tested preclinically and there are ongoing clinical trials for protein replacement purposes; however, more effort has been put into the development of prevention strategies against infectious diseases. Apparently, mRNA vaccine approval against coronavirus disease 2019 (COVID-19) is a landmark for opening new opportunities for managing diverse health disorders based on this approach. Indeed, apart from infectious diseases, it has also been widely tested in numerous directions including cancer prevention and the treatment of inherited disorders. Interestingly, self-amplifying RNA (saRNA)-based technology is believed to display more developed RNA therapy compared with conventional mRNA technique in terms of its lower dosage requirements, relatively fewer side effects, and possessing long-lasting effects. Nevertheless, some challenges still exist that need to be overcome in order to achieve saRNA-based drug approval in clinics. Hence, the current review discusses the feasibility of saRNA utility for protein replacement therapy on various health disorders including rare hereditary diseases and also provides a detailed overview of saRNA advantages, its molecular structure, mechanism of action, and relevant delivery platforms.     

辛德毕斯病毒载体的研究进展

辛德毕斯病毒(Sindbis virus,SINV)是一种具有广泛宿主嗜性的正链RNA病毒,能够在许多脊椎动物和无脊椎动物细胞中有效复制。其单股基因组RNA被分成两个开放阅读框(ORF),第1个ORF编码非结构蛋白,负责病毒RNA的转录和复制;第2个ORF在亚基因组启动子控制下编码病毒结构蛋白,负责病毒RNA的包裹和病毒粒子的组装。近年来,许多基于SINV的复制子载体已被开发,这些载体能够自我复制,但只有在与表达结构蛋白的辅助RNA共转染时,才能包装成病毒样粒子。目前,SINV复制子载体已广泛应用于疫苗开发和癌症基因治疗,能够激发有效的体液免疫和细胞免疫反应。本文就SINV载体的研究进展作一综述。