Synthetic Oligonucleotides Research Articles

Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. We assessed the analytical performance of the assays using synthetic oligonucleotides, which were further validated on plasma cfDNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cfDNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring.

Synthesis and cloning of long repeat sequences using single-stranded circular DNA.

Non-coding repeat expansion causes several neurodegenerative diseases, such as fragile X syndrome, amyotrophic lateral sclerosis/frontotemporal dementia, and spinocerebellar ataxia (SCA31). Such repetitive sequences must be investigated to understand disease mechanisms and prevent them, using novel approaches. However, synthesizing repeat sequences from synthetic oligonucleotides is challenging as they are unstable, lack unique sequences, and exhibit propensity to make secondary structures. Synthesizing long repeat sequence using polymerase chain reaction is often difficult due to lack of unique sequence. Here, we employed a rolling circle amplification technique to obtain seamless long repeat sequences using tiny synthetic single-stranded circular DNA as template. We obtained 2.5-3kbp uninterrupted TGGAA repeats, which is observed in SCA31, and confirmed it using restriction digestion, Sanger and Nanopore sequencing. This cell-free, in vitro cloning method may be applicable for other repeat expansion diseases and be used to produce animal and cell culture models to study repeat expansion diseases in vivo and in vitro.

Thymine DNA glycosylase is an RNA-binding protein with high selectivity for G-rich sequences

Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG's affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA.

Acoustic Force Elastography Microscopy.

Hydrogel scaffolds have attracted attention to develop cellular therapy and tissue engineering platforms for regenerative medicine applications. Among factors, local mechanical properties of scaffolds drive the functionalities of cell niche. Dynamic mechanical analysis (DMA), the standard method to characterize mechanical properties of hydrogels, restricts development in tissue engineering because the measurement provides a single elasticity value for the sample, requires direct contact, and represents a destructive evaluation preventing longitudinal studies on the same sample. We propose a novel technique, acoustic force elastography microscopy (AFEM), to evaluate elastic properties of tissue engineering scaffolds. AFEM can resolve localized and two-dimensional (2D) elastic properties of both transparent and opaque materials with advantages of being non-contact and non-destructive. Gelatin hydrogels, neat synthetic oligo[poly(ethylene glycol)fumarate] (OPF) scaffolds, OPF hydroxyapatite nanocomposite scaffolds and ex vivo biological tissue were examined with AFEM to evaluate the elastic modulus. These measurements of Young's modulus range from approximately 2 kPa to over 100 kPa were evaluated and are in good agreement with finite element simulations, surface wave measurements, and DMA tests. The AFEM can resolve localized and 2D elastic properties of hydrogels, scaffolds and thin biological tissues. These materials can either be transparent or non-transparent and their evaluation can be done in a non-contact and non-destructive manner, thereby facilitating longitudinal evaluation. AFEM is a promising technique to quantify elastic properties of scaffolds for tissue engineering and will be applied to provide new insights for exploring elastic changes of cell-laden scaffolds for tissue engineering and material science.

Pressure-Enabled Drug Delivery (PEDD) of a class C TLR9 agonist in combination with checkpoint inhibitor therapy in a murine pancreatic cancer model.

729 Background: Systemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma (PDAC). This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent pre-clinical cancer models and early-phase clinical trials have demonstrated the efficacy of toll-like receptor 9 agonists (TLR9a), including the synthetic CpG oligonucleotide SD101, to stimulate innate and adaptive immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure Enabled Drug Delivery (PEDD) via Pancreatic Retrograde Venous Infusion (PRVI) of a TLR9a would improve responsiveness to systemic anti-programmed death receptor-1 (PD-1) checkpoint inhibitor therapy in a murine orthotopic PDAC model. Methods: Murine PDAC (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to PRVI saline (pSAL, n=9), systemic anti-PD1 100 mcg/mouse on Day 0, 2 and 4 (sAPD1, n = 6), 30mcg PRVI TLR9a (pTLR9a, n=9), 30mcg systemic TLR9a (sTLR9a, n=7) on Day 0, and combination 30mcg PRVI TLR9a on Day 0 and systemic anti-PD1 100 mcg/mouse on Day 0, 2 and 4 (COMBO, n =9). Fluorescently-labeled TLR9a (radiant efficiency [RE]) was measured on day 1. Blood and tumors were collected at necropsy 12 days after infusion. Results: All mice survived to necropsy. Site of tumor fluorescence measurements revealed higher intensity fluorescence in pTLR9a compared to sTLR9a (7.5x10^5 vs. 2.4x10^5 RE, p= 0.048). Significantly lower MDSCs in the COMBO vs. pSAL are shown. Tumor weights were significantly lower in the COMBO group compared to pSAL (400 vs. 964 mg, p=0.003), and were also lower, although not significantly, than in the pTLR9a (400 vs. 518mg, p=0.50), and sAPD1 (400 vs. 645 mg, p = 0.70) groups. Conclusions: PEDD of TLR9a by PRVI with systemic anti-PD-1 demonstrated improved PDAC tumor control. These results support study of this combination therapy in PDAC patients and expansion of ongoing PEDD clinical trials. [Table: see text]

Preparation of a Convertible Spacer Containing a Disulfide Group for Versatile Functionalization of Oligonucleotides

The protocols described in this article provide details regarding the synthesis and characterization of a disulfide containing linker phosphoramidite for terminal functionalization of synthetic oligonucleotides. The linker is first synthesized from 6-mercaptohexanol in two steps and is incorporated at the 5' end of short DNA oligonucleotides using automated solid-phase synthesis. The linker contains a terminal tosylate group which is post-synthetically displaced by altering the deprotection conditions to yield a variety of functional handles (N3 , NH2 , OMe, SH) or alternatively, the tosylate can be displaced directly with primary amines such as tert-butylamine. The linker system is also compatible with RNA oligonucleotides enabling the introduction of various functional handles (N3 , NH2 ). The protocol outlined in this procedure provides access to a versatile linker for the terminal functionalization of oligonucleotides containing a disulfide bond which may serve useful in the synthesis of reduction-responsive oligonucleotide conjugates. As a proof of concept, in this protocol the linker is used to modify a dT10 oligonucleotide and then conjugated by copper(I)-mediated azide-alkyne cycloaddition (CuAAC) to an alkyne-modified poly(ethylene glycol) which shows concentration dependent release of the oligonucleotide upon treatment with 1,4-dithiothreitol, a reducing agent. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of disulfide linker phosphoramidite 3 Basic Protocol 2: Synthesis, functionalization, and characterization of DNA oligonucleotides containing disulfide linker phosphoramidite 3 Basic Protocol 3: Displacement of terminal tosylate functionalized DNA with primary aliphatic amines Basic Protocol 4: Synthesis of oligonucleotide-PEG conjugate Support Protocol: Preparation of PEG-alkyne.

Thermodynamics and kinetics of the B-S transition in base-modified DNA.

Understanding changes in the mechanical properties of nucleic acids is important to understand many biological processes. DNA exhibits a conformational change from canonical B- to overstretched S-form, characterized by 70% elongation, at ∼60–70 pN. The structure of S-form DNA is still debated and also difficult to distinguish from force-induced melting and peeling of DNA. Here, we characterized the kinetics and thermodynamics of the B-S transition and also investigated the effect of base modifications on the transition using tricyclic cytosine tC, which forms hydrogen bonds with guanine and changes the base stacking properties of B-DNA. The B-S transition in 60 bp DNA occurs at ∼63.5 pN (at 1 M salt) and fraying of ∼20 bp precedes the cooperative B-to-S transition of the remaining ∼40 bp. The B-S transition force increases gradually with tC incorporation, whereas the extension and free energy of the transition decrease. We conclude that fewer bp undergo the cooperative B-S transition in tC-stabilized duplexes, due to more fraying of B-DNA at higher transition forces, consistent with the transition becoming faster. For the first time we characterize the kinetics of the cooperative B-to-S transition and it occurs significantly faster than the alternative melting transition, while the extension of B-DNA at the B-to-S transition barrier is almost half-way between B- and S-DNA, suggesting an elastic nature of this transition. By characterizing the free energies of tC-modified relative to unmodified oligos, we estimated that each tC stabilizes the duplex by ∼3 kBT. The approach of studying short synthetic oligonucleotides with modified bases opens up new opportunities for understanding the local structure of nucleic acids with base-pair resolution.

SELECTION OF SPECIFIC PRIMERS FOR PCR IN BOVINE LEUKEMIA VIRUS

the polymerase chain reaction (PCR) method is widely used to solve various problems. PCR is widely used for the detection of bacterial and viral pathogens. Primers are a very important component of PCR, since the specificity of amplification depends primarily on them. They are necessary for the enzyme to work and are specific to the fragment of interest. Based on the results of the selection of nucleotide sequences of the genome or individual fragments of the virus RNA from the international database on the NCBI website, a large number of sequences for the bovine leukemia virus were identified, which are stored in gene banks and updated daily with new data. The construction of primers in compliance with the necessary parameters is carried out using various computer programs, the main of which are MUSCLE, UGENE V.36.0, Primer-BLAST, Oligo Analyzer and others. The designed primers were then synthesized on the Expedite 8909 oligonucleotide synthesizer, according to the instructions attached to the device. As a result of our experiments, we selected and synthesized specific synthetic oligonucleotides env (g51)_1 and env (g51)_2, for setting up PCR for bovine leukemia.

Molecular elements: novel approaches for molecular building.

Classically, a molecular element (ME) is a pure substance composed of two or more atoms of the same element. However, MEs, in the context of this review, can be any molecules as elements bonded together into the backbone of synthetic oligonucleotides (ONs) with designed sequences and functions, including natural A, T, C, G, U, and unnatural bases. The use of MEs can facilitate the synthesis of designer molecules and smart materials. In particular, we discuss the landmarks associated with DNA structure and related technologies, as well as the extensive application of ONs, the ideal type of molecules for intervention therapy aimed at correcting disease-causing genetic errors (indels). It is herein concluded that the discovery of ON therapeutics and the fabrication of designer molecules or nanostructures depend on the ME concept that we previously published. Accordingly, ME will be our focal point as we discuss related research directions and perspectives in making molecules and materials. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

The Pro-Oxidant Effect of Class A CpG ODNs on Human Neutrophils Includes Both Non-Specific Stimulation of ROS Production and Structurally Determined Induction of NO Synthesis

Synthetic CpG oligonucleotides are promising components of immunomodulatory drugs for the treatment and prophylaxis of infectious diseases, cancers, and allergies. Phosphorothioate modification stabilizes these compounds, contributing to the achievement of a clinical effect, but at the same time changes their immunomodulatory properties. We used the diffusible fluorescent dye dihydroethidium and the non-diffusible 6-carboxy-2′,7′dihydrochlorofluorescein diacetate and cytochrome c probes to demonstrate that it is the phosphorothioate backbones that determine the pronounced nonspecific pro-oxidant effect of CpG ODN on neutrophils. At the same time, as was shown using diaminofluorescein diacetate, the potentiation of nitric oxide synthesis in these leucocytes by CpG ODN class A strictly depends on the presence of CpG motifs and a palindromic “hairpin”. The results obtained will contribute to a more complete understanding of the physiological action of therapeutic agents based on synthetic CpG oligonucleotides.

Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms.

MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Two-tailed reverse transcription of A-to-I-edited microRNAs Basic Protocol 2: SYBR Green-based qPCR for A-to-I-edited microRNAs Alternate Protocol: Hydrolysis probe-based qPCR for A-to-I-edited microRNAs Support Protocol: Preparation of standard curves using synthetic RNA oligonucleotides.

Identification of Ribosomal Footprints on the Electrophoretic Gel in Translatome Profiling: on the Use of DNA Size Standards

The commercial DNA ladder was tested as a substitute for RNA size standards to identify ribosomal footprints (RNA fragments of about 30 nucleotides long) on an electrophoretic polyacrylamide gel for the purposes of translatome profiling. It has been found that 25 and 35 nucleotides long synthetic RNA oligonucleotides do migrate slower than the synthetic DNA oligonucleotides of the matching length and sequences and their positions on the gel coincide with those of 30 and 40 nucleotides long DNA oligonucleotides, correspondingly, of the commercial IDT 20/100 DNA oligo length standards. By using this DNA ladder and RNA isolated from the preparation enriched in ribosomes (obtained by fractionating on MicroSpin S-400 columns the HepG2 cell lysate treated with RNase I), the position of a band of putative ribosomal footprints can be identified on a gel that has been verified by measuring in an RNA-seq experiment the length of RNA fragments extracted from the band.

Selecting Therapeutic Antisense Oligonucleotides with Gene Targeting and TLR8 Potentiating Bifunctionality.

A growing body of preclinical evidence has led to the hypothesis that human Toll-like receptor 8 (hTLR8) activation in the tumor microenvironment (TME) could have potent anticancer effects through its action on monocytes, myeloid dendritic cells (mDCs), and natural killer (NK) cells. This has motivated the initiation of several clinical trials for chemical hTLR8 agonists in a variety of cancers. Concurrently, a growing number of synthetic antisense oligonucleotides (ASOs) are being developed as cancer therapeutics. We have recently reported that 2'-O-methyl (2'OMe)-modified ASOs can potentiate sensing of hTLR8 chemical agonists in a sequence-dependent manner. This suggests that select gene-targeting ASOs with anticancer activity may synergize with low-dose hTLR8 agonists in the TME. Here, we provide a detailed protocol to rapidly screen and identify such synthetic bifunctional oligonucleotides with synergistic activity on hTLR8 sensing.

A DNA Cleavage Assay Using Synthetic Oligonucleotide Containing a Single Site-Directed Lesion for In Vitro Base Excision Repair Study.

Many chemicals cause mutation or cancer in animals and humans by forming DNA lesions, including base adducts, which play a critical role in mutagenesis and carcinogenesis. A large number of such adducts are repaired by the DNA glycosylase-mediated base excision repair (BER) pathway, and some are processed by nucleotide excision repair (NER) and nucleotide incision repair (NIR). To understand what structural features determine repair enzyme specificity and mechanism in chemically modified DNA in vitro, we developed and optimized a DNA cleavage assay using defined oligonucleotides containing a single, site specifically placed lesion. This assay can be used to investigate novel activities against any newly identified derivatives from chemical compounds, substrate specificity and cleavage efficiency of repair enzymes, and quantitative structure-function relationships. Overall, the methodology is highly sensitive and can also be modified to explore whether a lesion is processed by NER or NIR activity, as well as to study its miscoding properties in translesion DNA synthesis (TLS).

Assay design for analysis of human uracil DNA glycosylase.

Human uracil DNA glycosylase (UNG2) is an enzyme whose primary function is to remove uracil bases from genomic DNA. UNG2 activity is critical when uracil bases are elevated in DNA during class switch recombination and somatic hypermutation, and additionally, UNG2 affects the efficacy of thymidylate synthase inhibitors that increase genomic uracil levels. Here, we summarize the enzymatic properties of UNG2 and its mitochondrial analog UNG1. To facilitate studies on the activity of these highly conserved proteins, we discuss three fluorescence-based enzyme assays that have informed much of our understanding on UNG2 function. The assays use synthetic DNA oligonucleotide substrates with uracil bases incorporated in the DNA, and the substrates can be single-stranded, double-stranded, or form other structures such as DNA hairpins or junctions. The fluorescence signal reporting uracil base excision by UNG2 is detected in different ways: (1) Excision of uracil from end-labeled oligonucleotides is measured by visualizing UNG2 reaction products with denaturing PAGE; (2) Uracil excision from dsDNA substrates is detected in solution by base pairing uracil with 2-aminopurine, whose intrinsic fluorescence is enhanced upon uracil excision; or (3) UNG2 excision of uracil from a hairpin molecular beacon substrate changes the structure of the substrate and turns on fluorescence by relieving a fluorescence quench. In addition to their utility in characterizing UNG2 properties, these assays are being adapted to discover inhibitors of the enzyme and to determine how protein-protein interactions affect UNG2 function.

RNA antisense and silencing strategies using synthetic drugs for rare muscular and neuromuscular diseases

Rare diseases occur in their large majority from a genetic cause, which makes them good candidates for genetic RNA drugs. The basic concepts, principles, mechanisms of action and chemical optimizations of synthetic antisense oligonucleotides (ASO) and small interfering RNA (siRNA) are illustrated. These drugs act either by leading to RNA degradation, or as steric blockers of RNA translation, microRNA antagonists, splicing modulators or inducers of exon skipping. Chemical modifications and delivery techniques differ and are adapted to their distinct functions. The successes, potential, and challenges of synthetic RNA drugs are illustrated for several muscular and neuromuscular diseases: Duchenne muscular dystrophy, spinal muscular atrophy, transthyretin amyloidosis, Type 1 myotonic dystrophy, centronuclear myopathy, oculopharyngeal muscular dystrophy.

Chromosome Painting Using Chromosome-Specific BAC Clones.

Chromosome painting (CP) refers to visualization of large chromosome regions, chromosome arms or entire chromosomes via fluorescence in situ hybridization (FISH) of chromosome-specific DNA sequences. For CP in crucifers (Brassicaceae), typically contigs of chromosome-specific bacterial artificial chromosomes (BAC) from Arabidopsis thaliana are applied as painting probes on chromosomes of A. thaliana or other species (comparative chromosome painting, CCP). CP/CCP enables to identify and trace particular chromosome regions and/or chromosomes throughout all mitotic and meiotic stages as well as corresponding interphase chromosome territories. However, extended pachytene chromosomes provide the highest resolution of CP/CCP. Fine-scale chromosome structure, structural chromosome rearrangements (such as inversions, translocations, centromere repositioning), and chromosome breakpoints can be investigated by CP/CCP. BAC DNA probes can be accompanied by other types of DNA probes, such as repetitive DNA, genomic DNA, or synthetic oligonucleotide probes. Here, we describe a robust step-by-step protocol of CP and CCP which proved to be efficient across the family Brassicaceae, but which is also applicable to other angiosperm families.

Chemical Synthesis of Oligonucelotide Sequences: Phosphoramidite Chemistry.

Oligonucleotides are used in a variety of molecular biology techniques, from next-generation sequencing to genetic testing. Maintaining the sequence fidelity of synthetic oligonucleotides is critical to their use. This chapter describes the steps of solid phase oligonucleotide synthesis and purification, which enables the synthesis of oligonucleotides with specific sequences and high purity.

5-Dihydroxyboryluridine enhances cytosolic penetration of antisense oligonucleotides.

The most significant challenge for nucleic acid drug development is their delivery across the cell membrane. Herein, we harness the reversible binding between boronic acids and cell surface glycans to aid in the cellular delivery of synthetic oligonucleotides. We install the artificial nucleotide 5-dihydroxyboryluridine (5boU) in a site-specific manner within druglike antisense oligonucleotides and demonstrate that these boronate-containing nucleic acids have enhanced cytosolic penetration and splice-correcting activity compared to non-boronate analogs. Strategic incorporation of 5boU is a simple, modular, and potentially general means of enhancing cellular delivery of therapeutic nucleic acids.

Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.

Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).