Unmodified DNA Oligonucleotides Research Articles

Detection of SARS-CoV-2 RNA Using a DNA Aptamer Mimic of Green Fluorescent Protein.

RNA detection is important in diverse diagnostic and analytical applications. RNAs can be rapidly detected using molecular beacons, which fluoresce upon hybridizing to a target RNA but require oligonucleotides with complex fluorescent dye and quencher conjugations. Here, we describe a simplified method for rapid fluorescence detection of a target RNA using simple unmodified DNA oligonucleotides. To detect RNA, we developed Lettuce, a fluorogenic DNA aptamer that binds and activates the fluorescence of DFHBI-1T, an otherwise nonfluorescent molecule that resembles the chromophore found in green fluorescent protein. Lettuce was selected from a randomized DNA library based on binding to DFHBI-agarose. We further show that Lettuce can be split into two separate oligonucleotide components, which are nonfluorescent on their own but become fluorescent when their proximity is induced by a target RNA. We designed several pairs of split Lettuce fragments that contain an additional 15-20 nucleotides that are complementary to adjacent regions of the SARS-CoV-2 RNA, resulting in Lettuce fluorescence only in the presence of the viral RNA. Overall, these studies describe a simplified RNA detection approach using fully unmodified DNA oligonucleotides that reconstitute the Lettuce aptamer templated by RNA.

Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA.

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

Application of Magnetic Nanoparticles Coated with Crosslinked Zwitterionic Poly(ionic liquid)s for the Extraction of Oligonucleotides.

Magnetic nanoparticles coated with zwitterionic poly(ionic liquid)s were applied for dispersive solid-phase extraction of oligonucleotides. The materials were synthesized by miniemulsion copolymerization of ionic liquids and divinylbenzene on magnetic nanoparticles. The functional monomers contain a positively charged imidazolium ring and one of the anionic groups: derivatives of acetate, malonate, or butyl sulfonate ions. Adsorption of unmodified DNA oligonucleotide on obtained materials was possible in ion-exchange (IE) and hydrophilic interactions (HI) mode. The adsorption in IE was possible at low pH and was almost complete. The adsorption in HI mode required the usage of appropriate addition of organic solvent but did not provide full adsorption. Studies on the desorption of the analytes included determining the impact of ammonium acetate concentration and pH and organic solvents addition on the recovery. The material containing acetic fragments as an anionic group was selected for the final procedure with the use of 10 mM ammonium acetate (pH = 9.5)/methanol (50/50, v/v) as an elution solution. The magnetic dispersive solid-phase extraction procedure was tested for the oligonucleotides with various modifications and lengths. Moreover, it was applied to extract DNA oligonucleotide and its synthetic metabolites from enriched human plasma without any pre-purification, with recoveries greater than 80%.

A sensitive ratiometric electrochemical biosensor based on DNA four-way junction formation and enzyme-assisted recycling amplification.

A simple ratiometric electrochemical biosensor is developed for sensitive detection of target DNA based on DNA four-way junction (DNA-4WJ) formation and enzyme-assisted recycling amplification. This biosensor can be easily fabricated by a one-step assembly of ratiometric probes and simply performed by a one-step incubation procedure. In the presence of target DNA, two unmodified DNA oligonucleotides may cooperatively hybridize with a hairpin probe in the triple-helix molecular beacon (THMB) to form a DNA-4WJ, which may cause conformational transduction and induce the change in the distance between two redox labeling probes and the electrode surface. The subsequent recognition and cleavage of DNA-4WJ quadripartite complexes by RNase HII may result in significant signal amplification. Due to the introduction of DNA-4WJ formation, enzyme-assisted recycling amplification and ratiometric measurement, this biosensor exhibits high sensitivity with a detection limit as low as 0.063 pM and a long dynamic range from 0.1 pM to 100 nM. Moreover, this biosensor demonstrates good performance with excellent selectivity, high reliability and good reproducibility, holding great potential for further applications in biomedical research and clinical diagnostics.

Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.

BackgroundA major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN).ResultsTargeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets.ConclusionWe find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.Electronic supplementary materialThe online version of this article (doi: 10.1186/1471-2164-15-401) contains supplementary material, which is available to authorized users.

The BCL2 Targeted Deoxyribonucleic Acid Inhibitor (DNAi) PNT2258 Is Active In Patients With Relapsed Or Refractory Non-Hodgkin's Lymphoma

BackgroundAberrant BCL2 control mechanisms are a defining characteristic of a variety of cancers including follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). PNT2258 is a 24-base, single-stranded, chemically unmodified phosphodiester DNA oligonucleotide encapsulated in a specialized liposome (SMARTICLES®) that has demonstrated antitumor activity against human cell lines and xenograft models that include NHL, prostate cancer and melanoma. The oligonucleotide sequence is designed to hybridize with genomic sequences that reside within 5'-untranscribed regulatory regions of the BCL2 gene to block transcription of BCL2 through a mechanism called DNA interference (DNAi). In a previously conducted phase I study, PNT2258 was safe and well tolerated at doses from 1 through 150 mg/m2 and PNT2258 showed evidence of BCL2-targeted effects on a variety of biomarkers. MethodsThis study is a multi-center, single-agent, pilot Phase II investigation of PNT2258 administered at a dose of 120 mg/m2 as a 3-hour IV infusion on days 1-5 of a 21-day cycle. Objectives included characterization of anti-tumor activity and safety data in patients (pts) with relapsed or refractory lymphoma undergoing treatment with PNT2258 for six cycles or until progressive disease or unacceptable toxicity. Adult pts with measureable disease that was FDG-PET positive at baseline were eligible. Additional criteria included ECOG PS ≤2, adequate bone marrow, renal and hepatic function, and prior treatment that included rituximab and cytotoxic chemotherapy. Standard CTCAE and Cheson criteria were used for evaluation. Patients were scanned with CT/PET at baseline, after 2 cycles and at study end. Details are at ClinicalTrials.gov under study number NCT01733238. ResultsTo date, 10 pts (median age, 61 [range 40 - 74]) have been treated with PNT2258. Median number of prior therapies was 4 (2 - 8) and 100% of pts had baseline adenopathy >5 cm with the majority >10 cm. Antitumor activity was observed in 4 of 4 patients with FL, including 1 complete response, 1 partial response (with 80% reduction in tumor size from 20 cm to 4 cm), and 2 patients with stable disease (SD); both with tumor shrinkage at interim (i.e., end-of-cycle 2) scan. One patient each with mantle cell lymphoma (MCL) and bulky chronic lymphocytic leukemia (CLL) has exhibited SD at interim scan. Two pts exhibited progressive disease (1 each MCL and CLL). Seven of ten pts continue treatment and updated results will be presented. Toxicity occurring in ≥2 pts included grade 1 chills (n=2), diarrhea (n=2), grade 2 nausea (n=3), tumor pain (n=2) and hypotension (n=2). The single grade 3 toxicity occurring in ≥2 pts was nausea (n=2) and no grade 4 toxicity was noted. Additionally, tumor lysis syndrome has not been observed. ConclusionsPNT2258 is a first-in-class DNAi consisting of a native, single-stranded, chemically unmodified DNA oligonucleotide delivered to a nuclear genomic target via a protective lipid nanoparticle, and the first to target the regulatory upstream region of the BCL2 gene. PNT2258 is well tolerated with an acceptable safety profile and shows significant single agent anti-tumor activity in pts with heavily treated relapsed NHL, particularly in pts with FL. Additional studies are planned. Disclosures:Gaylor:ProNAi Therapeutics, Inc.: Employment. Woolliscroft:ProNAi Therapeutics, Inc.: Employment. Rodrigueza:ProNAi Therapeutics, Inc.: Employment, Equity Ownership. Sooch:ProNAi Therapeutics, Inc.: Employment, Equity Ownership. Messmann:ProNAi Therapeutics, Inc.: Employment. Al-Katib:ProNAi Therapeutics, Inc.: Research Funding.

Development of A Bioanalytical Method For Quantification of A 15-Mer Oligonucleotide At Sub-Ng/Ml Concentrations Using Lc–MS/MS

LC-MS/MS provides a powerful technique for the selective quantification of therapeutic oligonucleotides; however, the LOQ (typically >1 ng/ml) may be higher than desirable for clinical bioanalysis. A method has been developed to allow quantification of a 15-mer unmodified DNA oligonucleotide in human plasma using SPE and UHPLC with MS/MS detection. The LOQ of this assay was 0.05 nM (∼250 pg/ml). This method was then further developed by the inclusion of online SPE to increase loading and apply additional sample cleanup. This allowed for improved assay precision at lower concentrations and increased signal, thus allowing the method to be validated over the range of 10-4000 pM (approximately 50-20,000 pg/ml). The method is accurate, precise and selective and it provides proof-of-concept for sub-ng/ml, high-throughput quantification of oligonucleotides using online SPE coupled to ion-pair, reversed-phase LC-MS/MS.

Abstract 2764: Effect of PNT2258 combinations with docetaxel, dacarbazine, or vemurafenib on the A375 melanoma xenograft

Abstract Bcl-2 and bcl-XL are anti-apoptotic proteins that promote drug resistance and enhance tumor cell survival. Bcl-2 gene amplification occurs in 90% of cutaneous melanomas implicating it as a potential target. PNT100 is a 24-mer unmodified DNA oligonucleotide complementary to a non-coding region of bcl-2. PNT2258, is comprised of PNT100 encapsulated in a unique pH tunable nuclear-targeted liposomes termed SMARTICLES®. Preliminary studies demonstrated fluorescently-labeled PNT100 in a SMARTICLE® is delivered to the cell nucleus and that PNT2258 produces dramatic tumor regression in a diffuse large cell lymphoma xenograft, prolonging survival with a coincident decrease in bcl-2 protein. PNT2258 activity in the A375 melanoma model was assessed as a single agent or in combination in two separate studies. Study 1: PNT2258 alone, IV, 5 days/week for 3 weeks, to mice bearing A375 produced growth delays of 10.4, 7.8, and 7.8 days at 12.5, 8.3, and 5.6 mg/kg/dose, respectively. Docetaxel alone, IV weekly x3, produced growth delays of 19.4 (1/10 TFS), 14.8 (1/10 TFS), and 11.5 days at 30, 20, and 13.3mg/kg/dose. Combination therapy produced growth delays of 45.3 (5/10 TFS), 18.9, 28.1 (2/10 TFS), and 25.0 (3/10 TFS) days at 12.5+30, 12.5+20, 5.6+30, and 5.6+20 mg/kg/dose, respectively. Study 2: PNT2258 alone produced a growth delay of 12 days at 15mg/kg/dose. Dacarbazine alone was ineffective at 80 mg/kg/dose IP. Combination therapy was not superior to PNT2258 alone. In contrast, vemurafenib alone (37.5 mg/kg/dose, PO, BIDx18 days) was highly active producing a growth delay in excess of 30 days, as did the combination of PNT2258+vemurafenib. PNT2258 is currently being evaluated for safety and tolerability in a phase Ia clinical trial. Preliminary mouse and human pharmacokinetics suggest that exposure levels in humans are similar to those producing anti-tumor efficacy in mice. Overall, 1) PNT2258+docetaxel produced a greater than additive effect compared to single agents, 2) PNT2258+dacarbazine was not superior to PNT2258 alone, 3) PNT2258+vemurafenib while highly active, was not superior to vemurafinib therapy alone, and 4) therapeutic PNT2258 exposures have been attained in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2764. doi:1538-7445.AM2012-2764

Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids – TINA

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5′ and 3′ termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch – in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

Enzyme-assisted binary probe for sensitive detection of RNA and DNA

The new enzyme-assisted assay for DNA/RNA detection provides real-time fluorescent signal readout along with low limit of detection and high discrimination power toward a single-base substitution. Requiring only two new unmodified DNA oligonucleotides for the detection of each new analyte, the assay is an efficient tool for low-cost analysis of multiple analytes.

LNA-modified oligonucleotides effectively drive intramolecular-stable hairpin to intermolecular-duplex state

Sequence-specific hybridization of antisense and antigene agent to the target nucleic acid is an important therapeutic strategy to modulate gene expression. However, efficiency of such agents falls due to inherent intramolecular-secondary-structures present in the target that pose competition to intermolecular hybridization by complementary antisense/antigene agent. Performance of these agents can be improved by employing structurally modified complementary oligonucleotides that efficiently hybridize to the target and force it to transit from an intramolecular-structured-state to an intermolecular-duplex state. In this study, the potential of variably substituted locked nucleic acid-modified oligonucleotides (8mer) to hybridize and disrupt highly stable, secondary structure of nucleic acid has been biophysically characterized and compared with the conventionally used unmodified DNA oligonucleotides. The target here is a stem–loop hairpin oligonucleotide—a structure commonly present in most structured-nucleic acids and known to exhibit an array of biological functions. Using fluorescence-based studies and EMSA we prove that LNA-modified oligonucleotides hybridize to the target hairpin with higher binding affinity even at lower concentration and subsequently, force it to assume a duplex conformation. LNA-modified oligonucleotides may thus, prove as potential therapeutic candidates to manipulate gene expression by disruption of biologically relevant nucleic acid secondary structure.