Preparing and Evaluating the Stability of Therapeutically Relevant Oligonucleotide Duplexes.

The field of oligonucleotide therapeutics is rapidly advancing, particularly for combating orphan diseases and cancer. However, the intrinsic instability of oligonucleotides, especially RNA, poses a substantial challenge in the face of the harsh conditions encountered intracellularly and in circulation. Therefore, evaluating the stability of oligos in serum is of great significance when developing oligonucleotide therapeutics. This protocol outlines a dependable and reproducible method for preparing oligonucleotide duplexes, coupled with confirmation by gel electrophoresis. Subsequently, the protocol defines a mechanism to assess the stability of the oligo duplexes in serum. This protocol seeks to establish a standardized reference for researchers, enabling them to compare the impact of various modifications on oligo stability and assess the degradation kinetics effectively. Key features • Adaptable for use with small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), and other unmodified and modified oligonucleotides. • Does not necessitate any Biological Safety Level clearance and offers a rapid, cost-effective, and entirely in vitro procedure. • Allows researchers to evaluate multiple modification patterns that, when coupled with targeting activity, allow for selecting the best modification pattern prior to in vivo analysis.

Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Engineering of functional nanosnowflakes from gold nanocarriers capped with amino-modified DNA oligonucleotides.

The design, engineering and electron microscopic characterization of anisotropic nanosized snowflake-like structural assemblies (nanosnowflakes) is reported. They were fabricated through immobilization of double stranded amine-modified and thiol-terminated DNA oligonucleotides on the surface of ultra-small isotropic gold nanoparticles used as nanocarriers. The transmission electron microscopy images combined with spectrophotometric data revealed the formation of self-assembled structural aggregation between individual ligands-coated nanoparticles. They act as seeds for the further spontaneous dendritic growth in different directions. Their anisotropic morphology is formed due to the occurrence of facilitated electrostatic interactions between positive charged amino-groups and the negative sugar-phosphate backbone of oligonucleotides. Thus, nanosnowflakes with size distribution between 40 and 80 nm were obtained. The microscopic analysis demonstrated also that the stable nanosnowflakes structure was highly dependent on the solution ionic strength, which effect the charge fluctuation within the assembly. The reported DNA functionalized nanostructures have potential to be applied as a platform for development of therapeutic materials, as well as drug delivery nanosystems. RESEARCH HIGHLIGHTS: The engineering, fabrication, and microscopic characterization of DNA nanosnowflakes is reported. The electron microscopy analysis revealed formation of self-assemblies with anisotropic morphology. The nanosnowflakes size distribution was between 40 and 80 nm.

Triazole-linked nucleic acids: Synthesis, therapeutics and synthetic biology applications.

This article covers the triazole-linked nucleic acids where the triazole linkage (TL) replaces the natural phosphate backbone. The replacement is done at either a few selected linkages or all the phosphate linkages. Two triazole linkages, the four-atom TL1 and the six-atom TL2, have been discussed in detail. These triazole-modified oligonucleotides have found a wide range of applications, from therapeutics to synthetic biology. For example, the triazole-linked oligonucleotides have been used in the antisense oligonucleotide (ASO), small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology as therapeutic agents. Due to the ease of the synthesis and a wide range of biocompatibility, the triazole linkage TL2 has been used to assemble a functional 300-mer DNA from alkyne- and azide-functionalized 100-mer oligonucleotides as well as an epigenetically modified variant of a 335 base-pair gene from ten short oligonucleotides. These outcomes highlight the potential of triazole-linked nucleic acids and open the doors for other TL designs and artificial backbones to fully exploit the vast potential of artificial nucleic acids in therapeutics, synthetic biology and biotechnology.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

Multi-Functionalized Heteroduplex Antisense Oligonucleotides for Targeted Intracellular Delivery and Gene Silencing in HeLa Cells.

Oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) are short synthetic nucleic acid molecules with a promising potential to treat a wide range of diseases. Despite considerable progress in the field, the development of safe and effective delivery systems that target organs and tissues other than the liver is challenging. While keeping possible off-target oligonucleotide interactions and toxicity related to chemical modifications in mind, innovative solutions for targeted intracellular delivery are highly needed. Herein, we report on the design, synthesis and testing of a novel multi-modified and multi-functionalized heteroduplex oligonucleotide (HDO) with respect to its intracellular delivery and its ability to silence genes in HeLa cells. Simultaneously, folic acid- and peptide- labeled HDO show proficient silencing of the green fluorescent protein (GFP) gene with an 84% reduction in the GFP fluorescence. In addition, the Bcl2 HDO achieved effective Bcl2 gene knockdown in the cells. The data show the proficiency of the multi-functionalization strategy and provide an example for advancing the design of safe and efficient forthcoming oligonucleotide therapeutics, such as HDO.

A reductionist approach to determine the effect of cell-cell contact on human epidermal stem cell differentiation.

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or 'niche'. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. STATEMENT OF SIGNIFICANCE: Stem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently.

Arabinonucleic acids containing C5-propynyl modifications form stable hybrid duplexes with RNA that are efficiently degraded by E. coli RNase H.

The promise of the antisense approach to treat a variety of diseases with oligonucleotides and solutions to challenges that have been encountered in their development is attributable to chemical modification of the nucleic acid scaffold. Herein, we describe preliminary data regarding the synthesis of a novel C5-propynyl-ß-d-arabinouridine (araUP) phosphoramidite and its incorporation into oligonucleotides. Substitution of araUP in dT18 results in minor stabilization of duplexes formed with RNA when modifications are placed consecutively and a uniformly modified araUP 18-mer increases stability by 34 °C relative to DNA. The modified oligomer exhibits improved nuclease and serum stability when compared to DNA and duplexes formed between RNA and araUP oligonucleotides are substrates for E. coli RNase H. These preliminary results merit further investigation into C5-propynyl modified arabino nucleic acids for potential therapeutic gene silencing applications.

Intrathecal Delivery of Therapeutic Oligonucleotides for Potent Modulation of Gene Expression in the Central Nervous System.

Therapeutic oligonucleotides hold tremendous potential for treating central nervous system (CNS) disorders. The route of administration of oligonucleotides significantly impacts both distribution and silencing efficiency. Here, we describe a technically simple, clinically relevant method to administer oligonucleotide compounds into the CNS via direct intrathecal injections. This method achieves distribution throughout the CNS rapidly and permits high-throughput testing of oligonucleotide efficacy and potency in mammals.

Triazole-Modified Nucleic Acids for the Application in Bioorganic and Medicinal Chemistry.

This review covers studies which exploit triazole-modified nucleic acids in the range of chemistry and biology to medicine. The 1,2,3-triazole unit, which is obtained via click chemistry approach, shows valuable and unique properties. For example, it does not occur in nature, constitutes an additional pharmacophore with attractive properties being resistant to hydrolysis and other reactions at physiological pH, exhibits biological activity (i.e., antibacterial, antitumor, and antiviral), and can be considered as a rigid mimetic of amide linkage. Herein, it is presented a whole area of useful artificial compounds, from the clickable monomers and dimers to modified oligonucleotides, in the field of nucleic acids sciences. Such modifications of internucleotide linkages are designed to increase the hybridization binding affinity toward native DNA or RNA, to enhance resistance to nucleases, and to improve ability to penetrate cell membranes. The insertion of an artificial backbone is used for understanding effects of chemically modified oligonucleotides, and their potential usefulness in therapeutic applications. We describe the state-of-the-art knowledge on their implications for synthetic genes and other large modified DNA and RNA constructs including non-coding RNAs.

Beyond ribose and phosphate: Selected nucleic acid modifications for structure-function investigations and therapeutic applications.

Over the past 25 years, the acceleration of achievements in the development of oligonucleotide-based therapeutics has resulted in numerous new drugs making it to the market for the treatment of various diseases. Oligonucleotides with alterations to their scaffold, prepared with modified nucleosides and solid-phase synthesis, have yielded molecules with interesting biophysical properties that bind to their targets and are tolerated by the cellular machinery to elicit a therapeutic outcome. Structural techniques, such as crystallography, have provided insights to rationalize numerous properties including binding affinity, nuclease stability, and trends observed in the gene silencing. In this review, we discuss the chemistry, biophysical, and structural properties of a number of chemically modified oligonucleotides that have been explored for gene silencing.

Synthetic Hexanucleotides as a Tool to Overcome Excessive Neutrophil Activation Caused by CpG-Containing Oligonucleotides.

Mimicking bacterial DNA, synthetic CpG-containing oligodeoxyribonucleotides (CpG-ODNs) have a powerful immunomodulatory potential. Their practical application is mainly associated with the production of vaccines, where they are used as adjuvants, as well as in local antimicrobial therapy. CpG-ODNs act on a wide variety of immune cells, including neutrophilic granulocytes. On the one hand, the stimulatory effect provides both the direct implementation of their antimicrobial and fungicidal mechanisms, and an avalanche-like strengthening of the immune signal due to interaction with other participants in the immune process. On the other hand, hyperactivation of neutrophilic granulocytes can have negative consequences. In particular, the formation of unreasonably high amounts of reactive oxygen species leads to tissue damages and, as a consequence, a spontaneous aggravation and prolongation of the inflammatory process. Under physiological conditions, a large number of DNA fragments are present in inflammation foci: both of microbial and self-tissue origin. We investigated effects of several short modified hexanucleotides on the main indicators of neutrophil activation, as well as their influence on the immunomodulatory activity of known synthetic CpG-ODNs. The results obtained show that short oligonucleotides partially inhibit the prooxidant effect of synthetic CpG-ODNs without significantly affecting the ability of the latter to overcome bacteria-induced pro-survival effects on neutrophilic granulocytes.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

Improved Cancer Targeting by Multimerizing Aptamers on Nanoscaffolds.

Aptamers are short single-stranded oligonucleotides selected to bind with high affinity and specificity to a target. In contrast to antibodies, aptamers can be produced in large-scale in vitro systems without the need for any biological agents, making them highly attractive as targeting ligands for bioimaging and drug delivery. For in vivo applications it is often desirable to multimerize the aptamers in order to increase their binding strength and overall specificity. Additional functionalities, such as imaging and therapeutic agents, as well as pharmacokinetic modifiers, need to be attached in a stoichiometric fashion. Herein, we present a robust method for assembly of up to three aptamers and a fluorophore in a single well-defined nanostructure. The process is entirely modular and can be applied to any aptamer requiring only a single reactive "click handle." Multimerization of two aptamers, A9g and GL21.T, previously shown to target cancer cells, led to a strong increase in cell uptake. A similar effect was observed for the prostate-specific membrane antigen (PSMA)-targeting A9g aptamer in mice where multivalent aptamer binding led to increased tumor specificity. Altogether, this method provides a platform for multimerization of aptamers with advantages in terms of combinatorial screening capacity and multifunctional design of nanomedicine.

Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

Synthesis of Oligonucleotides Containing 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) Moieties Using Post-Synthetic Modification Strategy.

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2'-urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2'-amino-LNA. Thus, 2'-urea-LNAs could be promising units for the modification of oligonucleotides; the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.

Inhibition of breast cancer cell proliferation with anti-microRNA oligonucleotides flanked by interstrand cross-linked duplexes.

Breast cancer is the most frequent cancer affecting women worldwide. Traditional chemotherapy, hormone therapy, and targeted therapy are used for breast cancer treatment. However, breast cancer is a heterogeneous disease, and patients often develop drug resistance. Therefore, various new therapeutic strategies have been investigated, including microRNA regulation. Anti-microRNA oligonucleotides (AMOs) are one of the most potent agents in oligonucleotide therapy. The inhibition activity of an AMO can be increased by flanking its single-stranded antisense sequence (the widely used structure for AMOs) with interstrand cross-linked duplexes (CLDs). An extrastable CLD improves nuclease resistance and stabilizes hybridization with a target. This study investigated the effects of anti-microRNA-21 (miR-21) AMO modified with CLDs on breast cancer cells without using reporter assay. The CLD-modified AMO suppressed breast cancer cell proliferation for a long duration compared to other types of AMOs. In addition, it expectedly up-regulated the miR-21-controlled expression of tumor suppressor genes. Therefore, an AMO flanked by CLDs can be a promising strategy for breast cancer treatment.

Efficient Epidermal Growth Factor Receptor Targeting Oligonucleotide as a Potential Molecule for Targeted Cancer Therapy.

Epidermal growth factor receptor (EGFR) is associated with the progression of a wide range of cancers including breast, glioma, lung, and liver cancer. The observation that EGFR inhibition can limit the growth of EGFR positive cancers has led to the development of various EGFR inhibitors including monoclonal antibodies and small-molecule inhibitors. However, the reported toxicity and drug resistance greatly compromised the clinical outcome of such inhibitors. As a type of chemical antibodies, nucleic acid aptamer provides an opportunity to overcome the obstacles faced by current EGFR inhibitors. In this study, we have developed and investigated the therapeutic potential of a 27mer aptamer CL-4RNV616 containing 2'-O-Methyl RNA and DNA nucleotides. Our results showed that CL-4RNV616 not only displayed enhanced stability in human serum, but also effectively recognized and inhibited the proliferation of EGFR positive Huh-7 liver cancer, MDA-MB-231 breast cancer, and U87MG glioblastoma cells, with an IC50 value of 258.9 nM, 413.7 nM, and 567.9 nM, respectively. Furthermore, TUNEL apoptosis assay revealed that CL-4RNV616 efficiently induced apoptosis of cancer cells. In addition, clinical breast cancer biopsy-based immunostaining assay demonstrated that CL-4RNV616 had a comparable detection efficacy for EGFR positive breast cancer with commonly used commercial antibodies. Based on the results, we firmly believe that CL-4RNV616 could be useful in the development of targeted cancer therapeutics and diagnostics.

BACE1 Inhibition Using 2'-OMePS Steric Blocking Antisense Oligonucleotides.

Amyloid beta-peptide is produced by the cleavage of amyloid precursor protein by two secretases, a ß-secretase, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and a γ-secretase. It has been hypothesised that partial inhibition of BACE1 in individuals with a high risk of developing Alzheimer's disease may be beneficial in preventing cognitive decline. In this study, we report the development of a novel antisense oligonucleotide (AO) that could efficiently downregulate the BACE1 transcript and partially inhibit BACE1 protein. We designed and synthesised a range of 2'-OMethyl-modified antisense oligonucleotides with a phosphorothioate backbone across various exons of the BACE1 transcript, of which AO2, targeting exon 2, efficiently downregulated BACE1 RNA expression by 90%. The sequence of AO2 was later synthesised with a phosphorodiamidate morpholino chemistry, which was found to be not as efficient at downregulating BACE1 expression as the 2'-OMethyl antisense oligonucleotides with a phosphorothioate backbone variant. AO2 also reduced BACE1 protein levels by 45%. In line with our results, we firmly believe that AO2 could be used as a potential preventative therapeutic strategy for Alzheimer's disease.

Novel Peptide Conjugates of Modified Oligonucleotides for Inhibition of Bacterial RNase P.

Novel alternatives to traditional antibiotics are now of great demand for the successful treatment of microbial infections. Here, we present the engineering and properties of new oligonucleotide inhibitors of RNase P, an essential bacterial enzyme. The series of 2'-O-methyl RNA (2'-OMe-RNA) and phosphoryl guanidine oligonucleotides were targeted to the substrate-binding region of M1 RNA subunit of the RNase P. Uniformly modified 2'-OMe RNA and selectively modified phosphoryl guanidine oligonucleotides possessed good stability in biological media and effectively inhibited RNase P. Their conjugates with transporting peptides were shown to penetrate bacterial cells (Escherichia coli and Acinetobacter baumannii) and inhibit bacterial growth.