Review of fragmentation of synthetic single-stranded oligonucleotides by tandem mass spectrometry from 2014 to 2022.

The fragmentation of oligonucleotides by mass spectrometry allows for the determination of their sequences. It is necessary to understand how oligonucleotides dissociate in the gas phase, which allows interpretation of data to obtain sequence information. Since 2014, a range of fragmentation mechanisms, including a novel internal rearrangement, have been proposed using different ion dissociation techniques. The recent publications have focused on the fragmentation of modified oligonucleotides such as locked nucleic acids, modified nucleobases (methylated, spacer, nebularine and aminopurine) and modification to the carbon 2'-position on the sugar ring; these modified oligonucleotides are of great interest as therapeutics. Comparisons of different dissociation techniques have been reported, including novel approaches such as plasma electron detachment dissociation and radical transfer dissociation. This review covers the period 2014-2022 and details the new knowledge gained with respect to oligonucleotide dissociation using tandem mass spectrometry (without priori sample digestion) during that time, with a specific focus on synthetic single-stranded oligonucleotides.

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.

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.

Silver or gold? A comparison of nanoparticle modified electrochemical genosensors based on cobalt porphyrin-DNA.

We applied a cobalt-porphyrin modified DNA as electrochemical marker, which was attached to nanoparticles, to detect specific DNA sequences. We compare the performance of gold and silver NPs in oligonucleotide sensors to determine if a change in metal will lead to either higher sensitivity or different selectivity, based on the redox behaviour of silver vs. gold. Surprisingly, we find that using either gold or silver NPs yields very similar overall performance. The electrochemical measurements of both types of sensors show the same redox behaviour which is dominated by the cobalt porphyrin, indicating that the electron pathway does not include the NP, but there is direct electron transfer between the porphyrin and the electrode. Both sensors show a linear response in the range of 5 × 10-17-1 × 10-16 M; the limit of detection (LOD) is 3.8 × 10-18 M for the AuNP sensor, and 5.0 × 10-18 M for the AgNP sensor, respectively, which corresponds to the detection of about 20-50 DNA molecules in the analyte. Overall, the silver system results in a better DNA economy and using cheaper starting materials for the NPs, thus shows better cost-effectivness and could be more suitable for the mass-production of highly sensitive DNA sensors.

A Porphyrin-DNA Chiroptical Molecular Ruler With Base Pair Resolution.

DNA-based molecular rulers enable scientists to determine important parameters across biology, from the measurement of protein binding interactions, to the study of membrane dynamics in cells. However, existing rulers can suffer from poor nanometre resolution due to the flexible nature of linkers used to tether to the DNA framework. We aimed to overcome this problem using zinc and free-base porphyrin chromophores attached via short and rigid acetylene linkers. This connectivity enables the distance and angle between the porphyrins to be fine-tuned along the DNA scaffold. The porphyrins undergo favorable energy transfer and chiral exciton coupling interactions to act as highly sensitive molecular ruler probes. To validate the system, we monitored the detection of small changes in DNA structure upon intercalation of ethidium bromide. CD spectroscopy showed the porphyrins undergo highly sensitive changes in excitation coupling to facilitate base pair resolution of the novel system.

Bacteria and nanosilver: the quest for optimal production.

Silver nanoparticles (AgNPs) have potential uses in many applications, but current chemical production methods are challenged by scalability, limited particle stability, and the use of hazardous chemicals. The biological processes present in bacteria to mitigate metallic contaminants in their environment present a potential solution to these challenges. Before commercial exploitation of this technology can be achieved, the quality of bacteriogenic AgNPs needs to be improved for certain applications. While the colloidal and morphological stabilities of biogenic AgNPs are widely regarded as superior to chemogenic particles, little control over the synthesis of particle morphologies has been achieved in biological systems. This article reviews a range of biosynthetic reaction conditions and how they affect AgNP formation in bacteria to understand which are most influential. While there remains uncertainty, some general trends are emerging: higher Ag+ concentrations result in higher AgNP production, up to a point at which the toxic effects begin to dominate; the optimal temperature appears to be heavily species-dependent and linked to the optimal growth temperature of the organism. However, hotter conditions generally favor higher production rates, while colder environments typically give greater shape diversity. Little attention has been paid to other potentially important growth conditions including halide concentrations, oxygen exposure, and irradiation with light. To fully exploit biosynthetic production routes as alternatives to chemical methods, hurdles remain with controlling particle morphologies and require further work to elucidate and harness them. By better understanding the factors influencing AgNP production, a foundation can be laid from which shape-controlled production can be achieved.

Self-Assembled Porphyrazine Nucleosides on DNA Templates: Highly Fluorescent Chromophore Arrays and Sizing Forensic Tandem Repeat Sequences.

The formation of chromophore arrays using a DNA templating approach leads to the creation of supramolecular assemblies, where the optical properties of the overall system can be fine-tuned to a large extent. In particular, porphyrin derivatives have been shown to be versatile building blocks; mostly covalent chemistry was used for embedding the units into DNA strands. Self-assembly of porphyrin modified nucleosides, on the other hand, has not been investigated as a simplified approach. We report on the synthesis of a magnesium(II) tetraaza porphine (MgTAP) coupled to deoxyuridine, and array formation on DNA templates which contain well-defined oligo(dA) segments showing strong fluorescence enhancement which is significantly larger than that with a Zn-porphyrin. The use of the deep-eutectic solvent glycholine is essential for successful assembly formation. The system allows for sizing of short tandem repeat markers with multiple adenosines, thus the concept could be adaptable to in vitro forensic DNA profiling with a suitable set of different chromophores on all nucleosides.

Approaching single DNA molecule detection with an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin DNA conjugates.

We describe an ultrasensitive electrochemical genosensor based on gold nanoparticles and cobalt-porphyrin functionalised ssDNA probes. The sensitivity at the attomolar concentration level arises from an increased density of redox labels on the electrode surface compared to sensors without NP modification. The electrode detects as few as 23 DNA molecules, approaching single molecule detection.

Easy-to-perform and cost-effective fabrication of continuous-flow reactors and their application for nanomaterials synthesis.

The translation of continuous-flow microreactor technology to the industrial environment has been limited by cost and complexity of the fabrication procedures and the requirement for specialised infrastructure. In the present study, we have developed a significantly more cost-effective and easy-to-perform fabrication method for the generation of optically transparent, continuous-flow reactors. The method combines 3D printing of master moulds with sealing of the PDMS channels' replica using a pressure-sensitive adhesive tape. Morphological characterisation of the 3D printed moulds was performed and reactors were fabricated with an approximately square-shaped cross-section of 1 mm2. Notably, they were tested for operation over a wide range of volumetric flow rates, up to 20 ml/min. Moreover, the fabrication time (i.e., from design to the finished product) was <1 day, at an average material cost of ∼£5. The flow reactors have been applied to the production of both inorganic nanoparticles (silver nanospheres) and organic vesicular systems (liposomes), and their performance compared with reactors produced using more laborious fabrication methods. Numerical simulations were performed to characterise the transport of fluids and chemical species within the devices. The developed fabrication method is suitable for scaled-up fabrication of continuous-flow reactors, with potential for application in biotechnology and nanomedicine.

Precision Templated Bottom-Up Multiprotein Nanoassembly through Defined Click Chemistry Linkage to DNA.

We demonstrate an approach that allows attachment of single-stranded DNA (ssDNA) to a defined residue in a protein of interest (POI) so as to provide optimal and well-defined multicomponent assemblies. Using an expanded genetic code system, azido-phenylalanine (azF) was incorporated at defined residue positions in each POI; copper-free click chemistry was used to attach exactly one ssDNA at precisely defined residues. By choosing an appropriate residue, ssDNA conjugation had minimal impact on protein function, even when attached close to active sites. The protein-ssDNA conjugates were used to (i) assemble double-stranded DNA systems with optimal communication (energy transfer) between normally separate groups and (ii) generate multicomponent systems on DNA origami tiles, including those with enhanced enzyme activity when bound to the tile. Our approach allows any potential protein to be simply engineered to attach ssDNA or related biomolecules, creating conjugates for designed and highly precise multiprotein nanoscale assembly with tailored functionality.

Nanoarchitectonics with Porphyrin Functionalized DNA.

DNA is well-known as bearer of the genetic code. Since its structure elucidation nearly seven decades ago by Watson, Crick, Wilkins, and Franklin, much has been learned about its detailed structure, function, and genetic coding. The development of automated solid-phase synthesis, and with it the availability of synthetic DNA with any desired sequence in lengths of up to hundreds of bases in the best case, has contributed much to the advancement of the field of DNA research. In addition, classic organic synthesis has allowed introduction of a very large number of modifications in the DNA in a sequence specific manner, which have initially been targeted at altering the biological function of DNA. However, in recent years DNA has become a very attractive scaffold in supramolecular chemistry, where DNA is taken out of its biological role and serves as both stick and glue molecule to assemble novel functional structures with nanometer precision. The attachment of functionalities to DNA has led to the creation of supramolecular systems with applications in light harvesting, energy and electron transfer, sensing, and catalysis. Functional DNA is clearly having a significant impact in the field of bioinspired nanosystems. Of particular interest is the use of porphyrins in supramolecular chemistry and bionanotechnology, because they are excellent functional groups due to their electronic properties that can be tailored through chemical modifications of the aromatic core or through insertion of almost any metal of the periodic table into the central cavity. The porphyrins can be attached either to the nucleobase, to the phosphate group, or to the ribose moiety. Additionally, noncovalent templating through Watson-Crick base pairing forms an alternative and attractive approach. With this, the combination of two seemingly simple molecules gives rise to a highly complex system with unprecedented possibilities for modulation of function, and with it applications, particularly when combined with other functional groups. Here, an overview is given on the developments of using porphyrin modified DNA for the construction of functional assemblies. Strategies for the synthesis and characterization are presented alongside selected applications where the porphyrin modification has proven to be particularly useful and superior to other modifiers but also has revealed its limitations. We also discuss implications on properties and behavior of the porphyrin-DNA, where similar issues could arise when using other hydrophobic and bulky substituents on DNA. This includes particularly problems regarding synthesis of the building blocks, DNA synthesis, yields, solubility, and intermolecular interactions.

Ion Channels Made from a Single Membrane-Spanning DNA Duplex.

Because of their hollow interior, transmembrane channels are capable of opening up pathways for ions across lipid membranes of living cells. Here, we demonstrate ion conduction induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, our duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, we elucidate the microscopic conductance pathway. Ions flow at the DNA-lipid interface as the lipid head groups tilt toward the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures, and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism realized here with the smallest possible DNA-based ion channel offers a route to design a new class of synthetic ion channels with maximum simplicity.

Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling.

Porphyrins were attached to LNA uridine building blocks via rigid 5-acetylene or more flexible propargyl-amide linkers and incorporated into DNA strands. The systems show a greatly increased thermodynamic stability when using as little as three porphyrins in a zipper arrangement. Thermodynamic analysis reveals clustering of the strands into more ordered duplexes with both greater negative ΔΔS and ΔΔH values, and less ordered duplexes with small positive ΔΔS differences, depending on the combination of linkers used. The exciton coupling between the porphyrins is dependent on the flanking DNA sequence in the single stranded form, and on the nature of the linker between the nucleobase and the porphyrin in the double stranded form; it is, however, also strongly influenced by intermolecular interactions. This system is suitable for the formation of stable helical chromophore arrays with sequence and structure dependent exciton coupling.

Retaining individualities: the photodynamics of self-ordering porphyrin assemblies.

The retention of photochemical properties of individual chromophores is a key feature of biological light harvesting complexes. This is achieved despite extensive aggregation of the chromophores, which in synthetic chromophore assemblies often yields a change in spectral characteristics. As an alternative approach towards mimicking biological light harvesting complexes, we report the synthesis of porphyrin assemblies which retained the photochemical properties of the individual chromophore units despite their substantial aggregation. These new materials highlight a new bottom-up approach towards the design and understanding of more complex biomimetic and naturally occurring biological systems.

Porphyrin-modified DNA as Construction Material in Supramolecular Chemistry and Nano-architectonics.

An overview is given on the developments of using porphyrin-modified DNA for the construction of functional assemblies. Strategies for the synthesis and characterisation are presented alongside selected applications where the porphyrin modification has proven to be particularly useful, but also revealed its limitations. We also discuss implications on property and behaviour of the porphyrin-DNA, where similar issues could arise using other hydrophobic and bulky substituents on DNA.

EIS-based biosensor for ultra-sensitive detection of TNF-α from non-diluted human serum.

Serum background is a critical issue for biosensor development as it interferes with the detection of target molecules and may give rise to false positive signal. We present here highly sensitive and selective TNF-α biosensor which is able to detect TNF-α from non-diluted human serum using magnetic bead coupled antibody and electrochemical impedance spectroscopy (EIS) techniques. The process is designed to detect TNF-α from human serum in three stages; (1) abundant protein backgrounds are depleted from the serum using magnetic bead coupled albumin and IgG antibodies, (2) after background depletion TNF-α is captured using magnetic bead coupled TNF-α antibody, and (3) the captured TNF-α is eluted from the magnetic beads and measured using EIS technique in which comb structured gold microelectrodes array (CSGM) is utilized to enhance the detection sensitivity. The system is able to achieve the limit of detection (LOD) at 1 pg/ml (57 fM) and a linear relationship between increasing TNF-α concentrations and charge-transfer resistance in a dynamic range of 1-1000 pg/ml.

A highly sensitive electrochemical genosensor based on Co-porphyrin-labelled DNA.

We report the use of Co-porphyrins as electrochemical tags for a highly sensitive and selective genosensor. An avian influenza virus-based DNA sequence characteristic of H5N1 was detected at femtomolar levels from competing non-complementary sequences through hybridisation with the labeled DNA.

Construction of DNA-polymer hybrids using intercalation interactions.

Reversible addition-fragmentation chain transfer (RAFT) polymerisation was used to produce a range of polymers terminated with an acridine group, which intercalates efficiently into dsDNA; the structure of the polymer determines the nature and strength of the interaction. Using a short 63 base pair dsDNA, discrete and well-defined DNA-polymer hybrid nanoparticles were formed, which were characterised by dynamic light scattering, small-angle X-ray scattering and atomic force microscopy.