Correcting PCR amplification errors in unique molecular identifiers to generate accurate numbers of sequencing molecules.

Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.

Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2.

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. The motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport, respectively, in the cilium. WDR34 (also known as DYNC2I2), a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome and asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases, making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34-knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity and Hedgehog signalling were also affected.

Molecular flexibility of DNA as a key determinant of RAD51 recruitment.

The timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By directly fitting a mechanistic model to our experimental data, we demonstrate that the RAD51 polymerisation rate positively correlates with the flexibility of DNA. Once the RAD51-DNA complex is formed, however, RAD51 remains stably bound independent of DNA flexibility, but rapidly dissociates from flexible DNA when RAD51 self-association is perturbed. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage.

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. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators-DPQ and DPPZ-and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.

"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

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. Transient electron spin resonance spectroscopy revealed that within these DNA-assembled pentacene stacks, SF evolves via a bound triplet pair quintet state, which subsequently converts into free triplets. SF evolution via a long-lived quintet state sets specific requirements on intermolecular coupling, rendering the quintet spectrum and its zero-field-splitting parameters highly sensitive to intermolecular geometry. We have found that the experimental spectra and zero-field-splitting parameters are consistent with a slight systematic strain relative to the MD-optimized geometry. Thus, the transient electron spin resonance analysis is a powerful tool to test and refine the MD-derived structure models. DNA-encoded assembly of coupled semiconductor molecules allows controlled construction of electronically functional structures, but brings with it significant dynamic and polar disorders. Our findings here of efficient SF through quintet states demonstrate that these conditions still allow efficient and controlled semiconductor operation and point toward future opportunities for constructing functional optoelectronic systems.

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. These modified oligonucleotides were recognised by SNM1A at >1000-fold lower concentrations than their fragment counterparts. The exonuclease SNM1A is a key enzyme involved in the repair of interstrand crosslinks, a highly cytotoxic form of DNA damage. However, SNM1A and other enzymes of this class are poorly understood, as there is a lack of tools available to facilitate their study. Our novel approach of incorporating functional fragments into oligonucleotides is broadly applicable to generating modified oligonucleotide structures with high affinity for DNA damage-repair enzymes.

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. Application of AAC chemistry to nucleic acids allows labeling, ligation, and cyclization of oligonucleotides efficiently and cost-effectively relative to previously used chemical and enzymatic techniques. In this review, we provide a guide to inexperienced and knowledgeable researchers approaching the field of click chemistry with nucleic acids. We discuss in detail the chemistry, the available modified-nucleosides, and applications of AAC reactions in nucleic acid chemistry and provide a critical view of the advantages, limitations, and open-questions within the field.

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. We provide methods to selectively modify the DNA component and/or the protein cargo of these materials, thereby greatly expanding the range of chemical functionalities available to these systems. We have used our methodology to construct DNFs bearing multiple surface aptamers and peptides capable of binding to cancer cells that overexpress the HER2 oncobiomarker, demonstrating their potential for diagnostic and therapeutic applications.

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. The PDIs are integrated within DNA chains using phosphoramidite coupling chemistry, allowing selection of the DNA sequence to either side, and specification of intermolecular DNA hybridization. In this way, we have developed a "toolbox" for construction of any stacking sequence of these semiconducting molecules. We have discovered that we need to use a full hierarchy of interactions: DNA guides the semiconductors into specified close proximity, hydrophobic-hydrophilic differentiation drives aggregation of the semiconductor moieties, and local geometry and electrostatic interactions define intermolecular positioning. As a result, the PDIs pack to give substantial intermolecular π wave function overlap, leading to an evolution of singlet excited states from localized excitons in the PDI monomer to excimers with wave functions delocalized over all five PDIs in the pentamer. This is accompanied by a change in the dominant triplet forming mechanism from localized spin-orbit charge transfer mediated intersystem crossing for the monomer toward a delocalized excimer process for the pentamer. Our modular DNA-based assembly reveals real opportunities for the rapid development of bespoke semiconductor architectures with molecule-by-molecule precision.

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 in vitro and in vivo 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. In particular, the oligonucleotide loading of SNAs has been determined to be a critical parameter in SNA design. In order to ensure the successful function of SNAs, the degree of oligonucleotide loading has to be quantitatively determined to confirm that a dense oligonucleotide shell has been achieved. However, this can be time-consuming and may lead to multiple syntheses being required to achieve the necessary degree of surface functionalization. In this work we show how this limitation can be overcome by introducing an oligonucleotide modification. By replacing the phosphodiester bond on the oligonucleotide backbone with a phosphorothioate bond, SNAs even with a low DNA loading showed remarkable stability in the presence of nucleases. Furthermore, these chemically modified SNAs exhibited high selectivity and specificity toward the detection of mRNA in cellulo.

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. In their DNA and 2'-OMe RNA formats these combination probes display high duplex stability and fluorescence when bound to complementary DNA and RNA.

Clinical presentation, diagnostic findings and outcome of dogs undergoing surgical resection for intracranial meningioma: 101 dogs.

BACKGROUND: Meningioma is the most common primary brain neoplasm in dogs. Further information is required regarding the expected long-term prognosis of dogs following the surgical resection of an intracranial meningioma together with the influence of adjunctive therapies. Whilst there have been several studies reporting the long-term outcome of intracranial meningioma resection following surgery alone, surgery with the use of an ultrasonic aspirator, surgery combined with radiotherapy and surgery combined with the addition of hydroxyurea, it is currently unclear which type of adjunctive therapy is associated with the most favourable outcomes. The objective of this study is to describe the presentation and outcome of dogs undergoing surgery for the resection of an intracranial meningioma and the effect of clinical factors, adjunctive therapies and meningioma histopathological subtype on the long-term outcome. RESULTS: A hundred and one dogs that had intracranial surgery for meningioma resection were investigated from four referral centres. 94% of dogs survived to hospital discharge with a median survival time of 386 days. Approximately 50% of dogs survived for less than a year, 25% survived between 1 and 2 years, 15% survived between 2 and 3 years and 10% survived for greater than 3 years following discharge from hospital. One or more adjunctive therapies were used in 75 dogs and the analysis of the data did not reveal a clear benefit of a specific type of adjunctive therapy. Those dogs that had a transfrontal approach had a significantly reduced survival time (MST 184 days) compared to those dogs that had a rostrotentorial approach (MST 646 days; p < 0.05). There was no association between meningioma subtype and survival time. CONCLUSIONS: This study did not identify a clear benefit of a specific type of adjunctive therapy on the survival time. Dogs that had a transfrontal approach had a significantly reduced survival time. Intracranial surgery for meningioma resection offers an excellent prognosis for survival to discharge from hospital with a median long term survival time of 386 days.

Strict conformational demands of RNA cleavage in bulge-loops created by peptidyl-oligonucleotide conjugates.

Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. 'Smart' supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-ß anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an 'in-line' attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.

2'-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis.

The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2-10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2' position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2'-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor.

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.

Chemically modified nucleic acids and DNA intercalators as tools for nanoparticle assembly.

The self-assembly of inorganic nanoparticles to larger structures is of great research interest as it allows the fabrication of novel materials with collective properties correlated to the nanoparticles' individual characteristics. Recently developed methods for controlling nanoparticle organisation have enabled the fabrication of a range of new materials. Amongst these, the assembly of nanoparticles using DNA has attracted significant attention due to the highly selective recognition between complementary DNA strands, DNA nanostructure versatility, and ease of DNA chemical modification. In this review we discuss the application of various chemical DNA modifications and molecular intercalators as tools for the manipulation of DNA-nanoparticle structures. In detail, we discuss how DNA modifications and small molecule intercalators have been employed in the chemical and photochemical DNA ligation in nanostructures; DNA rotaxanes and catenanes associated with reconfigurable nanoparticle assemblies; and DNA backbone modifications including locked nucleic acids, peptide nucleic acids and borane nucleic acids, which affect the stability of nanostructures in complex environments. We conclude by highlighting the importance of maximising the synergy between the communities of DNA chemistry and nanoparticle self-assembly with the aim to enrich the library of tools available for the manipulation of nanostructures.

A Click Chemistry Approach to Targeted DNA Crosslinking with cis-Platinum(II)-Modified Triplex-Forming Oligonucleotides.

Limitations of clinical platinum(II) therapeutics include systemic toxicity and inherent resistance. Modern approaches, therefore, seek new ways to deliver active platinum(II) to discrete nucleic acid targets. In the field of antigene therapy, triplex-forming oligonucleotides (TFOs) have attracted interest for their ability to specifically recognise extended duplex DNA targets. Here, we report a click chemistry based approach that combines alkyne-modified TFOs with azide-bearing cis-platinum(II) complexes-based on cisplatin, oxaliplatin, and carboplatin motifs-to generate a library of PtII -TFO hybrids. These constructs can be assembled modularly and enable directed platinum(II) crosslinking to purine nucleobases on the target sequence under the guidance of the TFO. By covalently incorporating modifications of thiazole orange-a known DNA-intercalating fluorophore-into PtII -TFOs constructs, enhanced target binding and discrimination between target and off-target sequences was achieved.

Engineering Enzyme-Cleavable Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers.

Oligonucleotides containing cleavable linkers have emerged as versatile tools to achieve stimulus-responsive and site-specific cleavage of DNA. However, the limitations of previously reported cleavable linkers including photolabile and disulfide linkers have restricted their applications in vivo. Inspired by the cathepsin B-sensitive dipeptide linkers in antibody-drug conjugates (ADCs) such as Adcetris, we have developed Val-Ala-02 and Val-Ala-Chalcone phosphoramidites for the automated synthesis of enzyme-cleavable oligonucleotides. Cathepsin B digests Val-Ala-02 and Val-Ala-Chalcone linkers efficiently, enabling cleavage of oligonucleotides into two components or release of small-molecule payloads. Based on the prior success of dipeptide linkers in ADCs, we believe that these dipeptide linker phosphoramidites will promote new clinical applications of therapeutic oligonucleotides.

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 (TL2) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL2 has superior replication kinetics to these analogues and is accurately replicated by polymerases. Derived structure-biocompatibility relationships show that linker length and the orientation of a hydrogen bond acceptor are critical and provide further guidance for the rational design of artificial biocompatible nucleic acid backbones.