Synthesis, Biophysical and Biological Evaluation of Splice-Switching Oligonucleotides with Multiple LNA-Phosphothiotriester Backbones.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2'--methyl phosphorothioate nucleotides for therapeutic applications.

Synthesis of -Alkyl-3-[2-oxoquinolin-1(2)-yl]propanoic Acid Derivatives and Related Compounds: Cytotoxicity and EGFR Inhibition of Some Propanamide Derivatives.

A series of 20 new structure-modified quinolin-2-one derivatives were prepared for biological evaluation. This was successfully achieved based on chemoselective reactions of heterocyclic amides with acrylic acid derivatives, which gave 3-[2-oxoquinolin-1-(2)-yl] propanoic acid derivatives (N-substitution via a unique behavior). The ester was reacted with hydrazine to afford the corresponding hydrazide.

Modular and automated synthesis of oligonucleotide-small molecule conjugates for cathepsin B mediated traceless release of payloads.

The reversible attachment of small molecules to oligonucleotides provides versatile tools for the development of improved oligonucleotide therapeutics. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement for traceless release of the payload . Herein, we describe a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB) for the automated synthesis of oligonucleotide-small molecule conjugates.

Synthesis and Properties of Oligonucleotides Containing LNA-Sulfamate and Sulfamide Backbone Linkages.

Oligonucleotides hold great promise as therapeutic agents but poor bioavailability limits their utility. Hence, new analogues with improved cell uptake are urgently needed. Here, we report the synthesis and physical study of reduced-charge oligonucleotides containing artificial LNA-sulfamate and sulfamide linkages combined with 2'-O-methyl sugars and phosphorothioate backbones.

Thiazole orange-carboplatin triplex-forming oligonucleotide (TFO) combination probes enhance targeted DNA crosslinking.

We report a new class of carboplatin-TFO hybrid that incorporates a bifunctional alkyne-amine nucleobase monomer called AP-C3-dT that enables dual 'click' platinum(ii) drug conjugation and thiazole orange fluorophore coupling. Thiazole orange enhances the binding of Pt(ii)-TFO hybrids and provides an intrinsic method for monitoring triplex formation. These hybrid constructs possess increased stabilisation and crosslinking properties in comparison to earlier Pt(ii)-TFOs, and demonstrate sequence-specific binding at neutral pH.

Single-cell RNA-sequence analysis of human bone marrow reveals new targets for isolation of skeletal stem cells using spherical nucleic acids.

There is a wealth of data indicating human bone marrow contains skeletal stem cells (SSC) with the capacity for osteogenic, chondrogenic and adipogenic differentiation. However, current methods to isolate SSCs are restricted by the lack of a defined marker, limiting understanding of SSC fate, immunophenotype, function and clinical application. The current study applied single-cell RNA-sequencing to profile human adult bone marrow populations from 11 donors and identified novel targets for SSC enrichment.

Two-Step Validation Approach for Tools To Study the DNA Repair Enzyme SNM1A.

We report a two-step validation approach to evaluate the suitability of metal-binding groups for targeting DNA damage-repair metalloenzymes using model enzyme SNM1A. A fragment-based screening approach was first used to identify metal-binding fragments suitable for targeting the enzyme. Effective fragments were then incorporated into oligonucleotides using the copper-catalysed azide-alkyne cycloaddition reaction.

Photogeneration of Spin Quintet Triplet-Triplet Excitations in DNA-Assembled Pentacene Stacks.

Singlet fission (SF), an exciton-doubling process observed in certain molecular semiconductors where two triplet excitons are generated from one singlet exciton, requires correctly tuned intermolecular coupling to allow separation of the two triplets to different molecular units. We explore this using DNA-encoded assembly of SF-capable pentacenes into discrete π-stacked constructs of defined size and geometry. Precise structural control is achieved via a combination of the DNA duplex formation between complementary single-stranded DNA and the local molecular geometry that directs the SF chromophores into a stable and predictable slip-stacked configuration, as confirmed by molecular dynamics (MD) modeling.

A new phosphoramidite enables orthogonal double labelling to form combination oligonucleotide probes.

Oligonucleotides labelled with thiazole orange intercalator and a reporter dye on the same thymine base have been synthesized. The key phosphoramidite (AP-C3 dT) contains an alkyne and amine, enabling dual orthogonal labelling of the nucleobase. Multiple monomers can be added to produce heavily functionalised oligonucleotides.

An LNA-amide modification that enhances the cell uptake and activity of phosphorothioate exon-skipping oligonucleotides.

Oligonucleotides that target mRNA have great promise as therapeutic agents for life-threatening conditions but suffer from poor bioavailability, hence high cost. As currently untreatable diseases come within the reach of oligonucleotide therapies, new analogues are urgently needed to address this. With this in mind we describe reduced-charge oligonucleotides containing artificial LNA-amide linkages with improved gymnotic cell uptake, RNA affinity, stability and potency.

A SARS-Cov-2 sensor based on upconversion nanoparticles and graphene oxide.

Since the beginning of the COVID-19 pandemic, there has been an increased need for the development of novel diagnostic solutions that can accurately and rapidly detect SARS-CoV-2 infection. In this work, we demonstrate the targeting of viral oligonucleotide markers within minutes without the requirement of a polymerase chain reaction (PCR) amplification step the use of oligonucleotide-coated upconversion nanoparticles (UCNPs) and graphene oxide (GO).

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications.

Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications.

An Investigation into the Resistance of Spherical Nucleic Acids against DNA Enzymatic Degradation.

Nanoparticles coated with oligonucleotides, also termed spherical nucleic acids (SNAs), are at the forefront of scientific research and have been applied and for sensing, gene regulation, and drug delivery. They demonstrate unique properties stemming from the three-dimensional shell of oligonucleotides and present high cellular uptake. However, their resistance to enzymatic degradation is highly dependent on their physicochemical characteristics.

Deoxyribonucleic Acid Encoded and Size-Defined π-Stacking of Perylene Diimides.

Natural photosystems use protein scaffolds to control intermolecular interactions that enable exciton flow, charge generation, and long-range charge separation. In contrast, there is limited structural control in current organic electronic devices such as OLEDs and solar cells. We report here the DNA-encoded assembly of π-conjugated perylene diimides (PDIs) with deterministic control over the number of electronically coupled molecules.

A New 1,5-Disubstituted Triazole DNA Backbone Mimic with Enhanced Polymerase Compatibility.

Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the RuAAC-catalyzed synthesis of a novel 1,5-disubstituted triazole (TL) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL has superior replication kinetics to these analogues and is accurately replicated by polymerases.

Expanding the chemical functionality of DNA nanomaterials generated by rolling circle amplification.

Rolling circle amplification (RCA) is a powerful tool for the construction of DNA nanomaterials such as hydrogels, high-performance scaffolds and DNA nanoflowers (DNFs), hybrid materials formed of DNA and magnesium pyrophosphate. Such DNA nanomaterials have great potential in therapeutics, imaging, protein immobilisation, and drug delivery, yet limited chemistry is available to expand their functionality. Here, we present orthogonal strategies to produce densely modified RCA products and DNFs.

Artificial nucleic acid backbones and their applications in therapeutics, synthetic biology and biotechnology.

The modification of DNA or RNA backbones is an emerging technology for therapeutic oligonucleotides, synthetic biology and biotechnology. Despite a plethora of reported artificial backbones, their vast potential is not fully utilised. Limited synthetic accessibility remains a major bottleneck for the wider application of backbone-modified oligonucleotides.

DNA Gold Nanoparticle Motors Demonstrate Processive Motion with Bursts of Speed Up to 50 nm Per Second.

Synthetic motors that consume chemical energy to produce mechanical work offer potential applications in many fields that span from computing to drug delivery and diagnostics. Among the various synthetic motors studied thus far, DNA-based machines offer the greatest programmability and have shown the ability to translocate micrometer-distances in an autonomous manner. DNA motors move by employing a burnt-bridge Brownian ratchet mechanism, where the DNA "legs" hybridize and then destroy complementary nucleic acids immobilized on a surface.

An In-labelled bis-ruthenium(ii) dipyridophenazine theranostic complex: mismatch DNA binding and selective radiotoxicity towards MMR-deficient cancer cells.

Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics.

Enrichment of Skeletal Stem Cells from Human Bone Marrow Using Spherical Nucleic Acids.

Human bone marrow (BM)-derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic, and chondrogenic lineages, enabling their application to clinical therapies. However, current methods to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers alone can isolate a homogeneous cell population with the ability to form bone, cartilage, and adipose tissue in humans.

A Hitchhiker's Guide to Click-Chemistry with Nucleic Acids.

Click chemistry is an immensely powerful technique for the fast and efficient covalent conjugation of molecular entities. Its broad scope has positively impacted on multiple scientific disciplines, and its implementation within the nucleic acid field has enabled researchers to generate a wide variety of tools with application in biology, biochemistry, and biotechnology. Azide-alkyne cycloadditions (AAC) are still the leading technology among click reactions due to the facile modification and incorporation of azide and alkyne groups within biological scaffolds.

"Split-and-Click" sgRNA.

CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. Solid-phase RNA synthesis can overcome these issues, but highly pure full-length sgRNA remains at the limits of current synthetic methods.

Development of Gene-Targeted Polypyridyl Triplex-Forming Oligonucleotide Hybrids.

In the field of nucleic acid therapy there is major interest in the development of libraries of DNA-reactive small molecules which are tethered to vectors that recognize and bind specific genes. This approach mimics enzymatic gene editors, such as ZFNs, TALENs and CRISPR-Cas, but overcomes the limitations imposed by the delivery of a large protein endonuclease which is required for DNA cleavage. Here, we introduce a chemistry-based DNA-cleavage system comprising an artificial metallo-nuclease (AMN) that oxidatively cuts DNA, and a triplex-forming oligonucleotide (TFO) that sequence-specifically recognises duplex DNA.