Search Results (61)

Real-time PCR (qPCR) is today’s definitive quantitative technology in molecular biology and diagnostics. Until 30 years ago, PCR product analyses were generally performed after amplification using gel-based methods. Quantification typically relied on visual inspection or densitometry of end-point products and was therefore relatively unreliable and poorly suited to high-throughput automation. To celebrate real-time PCR’s 30-year anniversary of commercial availability, Professor Stephen Bustin, Guest Editor for the special edition, “Advancing Molecular Science Through Reproducible qPCR: MIQE Guidelines and Beyond,” asked Russell Higuchi to give a historical account on how his idea of real-time PCR was conceived and brought to fruition. Dr. Higuchi then asked his collaborator, Lincoln McBride, who drove the development of the ABI 7700—the high-throughput real-time PCR instrument that gave researchers access to this technology—to co-author this dual memoir. This story is told from the perspectives of the two scientists most directly responsible for making real-time PCR practical and widely accessible. Taking turns, Russell Higuchi describes the conceptual and experimental steps at Cetus and then Roche that led from homogeneous PCR detection to continuous fluorescence monitoring, whilst Lincoln McBride details ABI’s parallel efforts to commercialize Russ’s invention. Together, they trace how experimental insight, engineering constraints, product development, and commercial decision-making shaped the Applied Biosystems 7700 Sequence Detection System and established real-time PCR as a practical and reliable quantitative technology. Their team’s efforts persevered through technological uncertainty and within a complex corporate collaboration. They share key historical documents in their original form. Their accounts show how the 7700 system emerged as the convergence of chemistry, optics, software, and product development. The eventual global reliance on real-time PCR during the COVID-19 pandemic demonstrated, at unprecedented scale, the profound and enduring impact of these early technical and organizational choices. Full article

Light-sensitive protecting chemical groups play an important role in the control of chemical reactions with high spatial and temporal resolution. Various forms of o-nitrobenzyl as a protecting compound are used for light-directed DNA synthesis. The suitability of a particular derivative for the application is defined by its photolytic efficiency, a characteristic, that is commonly extracted from a repetitive HPLC procedure. Here, using an example of a phosphoramidite compound with improved properties of deprotection, we delineate a simplified and economic approach based on a measurement of absorbance spectra to evaluate the photolytic efficiency. The obtained values are in close agreement with those determined previously. Full article

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry. Full article

To develop a modifier based on diethanolamine, a corresponding phosphoramidite for automated solid-phase deoxyribonucleic acid synthesis was synthesized. The influence of this modifier on the thermal stability of the terminally modified nucleic acids showed a dependence on the neighboring nucleobases and could be attributed to the fraying of the DNA ends. The potential for modification with dioxazaborocanes was first investigated using a small molecule model, and the formation of the dioxazaborocane was confirmed both in solution and in the solid state. Such a modification could expand the scope of xenonucleic acids in the future and modulate the properties of nucleic acids in solution. The influence on the thermal stability of the modified nucleic acids was minimal. In the future, this modification will be extended to internal incorporation and the potential of dioxazaborocanes in the nucleic acid context will be further exploited. Full article

Aptasensors are biosensors that rely on an aptamer’s ability to selectively bind targets. To produce a signal indicative of successful binding, aptamers are frequently modified with reporter agents. However, modification of aptamers with specific reporter agents affects subsequent aptamer-target binding, resulting in time-consuming screening assays to identify suitable aptamers capable of binding, once modified. To address this, this study proposes a SELEX approach that amplifies an enriched aptamer pool using a 5′-biotin-C6-phosphoramidite modification and using that for subsequent selection of suitable sequences capable of binding when modified. For this study, fractions from an existing enriched aptamer pool from a previous SELEX for hCG aptamers were separately amplified utilising biotinylated and non-biotinylated (unmodified) primers and sequenced via nanopore next-generation sequencing. While several enriched sequences were represented within the pools, bioinformatic analysis of the pools indicated subtle clustering of sequences between pools. However, the disparity in the number of sequences between both pools may indicate a possible amplification or sequencing-based bias caused by the biotinylation. This approach has merit to support aptamer SELEX strategies but may require further validation. Full article

With the goal of fast and accurate diagnosis of infectious diseases, this study presents a novel electrochemical biosensor that employs a refined aptamer (C9t) for the detection of spike (S) protein SARS-CoV-2 variants in a flexible multielectrode aptasensor array with PoC capabilities. Two aptamer modifications were employed: removing the primer binding sites and including two dithiol phosphoramidite anchor molecules. Thus, reducing fabrication time from 24 to 3 h and increasing the stability and sparseness for multi-thiol aptasensors compared to a standard aptasensor using single thiols, without a reduction in aptamer density. The biosensor fabrication, optimization, and detection were verified in detail by electrochemistry, QCM-D, SPR, and XPS. The analyte–receptor binding was further confirmed spectroscopically at the level of individual molecules by AFM-IR. The aptasensor possesses a low limit of detection (8.0 fg/mL), the highest sensitivity reported for S protein (209.5 signal per concentration decade), and a wide dynamic detection range (8.0 fg/mL–38 ng/mL) in nasopharyngeal samples, covering the clinically relevant range. Furthermore, the C9t aptasensor showed high selectivity for SARS-CoV-2 S proteins over biomarkers for MERS-CoV, RSV, and Influenza. Even more, it showed a three times higher sensitivity for the Omicron in comparison to the Wuhan strain (wild type), alpha, and beta variants of the SARS-CoV-2 virus. Those results demonstrate the creation of an affordable and variant-selective refined C9t aptasensor that outperformed current rapid diagnosis tests. Full article

Nowadays, nucleic acid derivatives capable of modulating gene expression at the RNA level have gained widespread recognition as promising therapeutic agents. A suitable degree of biological stability of oligonucleotide therapeutics is required for in vivo application; this can be most expeditiously achieved by the chemical modification of the internucleotidic phosphate group, which may also affect their cellular uptake, tissue distribution and pharmacokinetics. Our group has previously developed a strategy for the chemical modification of the phosphate group via the Staudinger reaction on a solid phase of the intermediate dinucleoside phosphite triester and a range of, preferably, electron deficient organic azides such as sulfonyl azides during automated solid-phase DNA synthesis according to the conventional β-cyanoethyl phosphoramidite scheme. Polyfluoro compounds are characterized by unique properties that have prompted their extensive application both in industry and in scientific research. We report herein the synthesis and isolation of novel oligodeoxyribonucleotides incorporating internucleotidic perfluoro-1-octanesulfonyl phosphoramidate or 2,2,2-trifluoroethanesulfonyl phosphoramidate groups. In addition, novel oligonucleotide derivatives with fluorinated zwitterionic phosphate mimics were synthesized by a tandem methodology, which involved (a) the introduction of a carboxylic ester group at the internucleotidic position via the Staudinger reaction with methyl 2,2-difluoro-3-azidosulfonylacetate; and (b) treatment with an aliphatic diamine, e.g., 1,1-dimethylethylenediamine or 1,3-diaminopropane. It was further shown that the polyfluoro oligonucleotides obtained were able to form complementary duplexes with either DNA or RNA, which were not significantly differing in stability from the natural counterparts. Long-chain perfluoroalkyl oligonucleotides were taken up into cultured human cells in the absence of a transfection agent. It may be concluded that the polyfluoro oligonucleotides described here can represent a useful platform for designing oligonucleotide therapeutics. Full article

N-Acetylgalactosamine (GalNAc) is an efficient and multifunctional delivery tool in the development and synthesis of chemically modified oligonucleotide therapeutics (conjugates). Such therapeutics demonstrate improved potency in vivo due to the selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis, which is what drives the high interest in this molecule. The ways to synthesize such structures are relatively new and have not been optimized in terms of the yields and stages both in lab and large-scale synthesis. Another significant criterion, especially in large-scale synthesis, is to match ecological norms and perform the synthesis in accordance with the Green Chemistry approach, i.e., to control and minimize the amounts of reagents and resources consumed and the waste generated. Here, we provide a robust and resource effective pot-economy method for the synthesis of triantennary GalNAc and GalNAc phosphoramidite/CPG optimized for laboratory scales. Full article

Data storage on DNA has emerged as a molecular approach to safeguarding digital information. Microarrays are an excellent source of complex DNA sequence libraries and are playing a central role in the development of this technology. However, the amount of DNA recovered from microarrays is often too small, and a PCR amplification step is usually required. Primer information can be conveyed alongside the DNA library itself in the form of readable barcodes made of DNA on the array surface. Here, we present a synthetic method to pattern QR and data matrix barcodes using DNA photolithography, phosphoramidite chemistry and fluorescent labeling. Patterning and DNA library synthesis occur simultaneously and on the same surface. We manipulate the chemical composition of the barcodes to make them indelible, erasable or hidden, and a simple chemical treatment under basic conditions can reveal or degrade the pattern. In doing so, information crucial to retrieval and amplification can be made available by the user at the appropriate stage. The code and its data contained within are intimately linked to the library as they are synthesized simultaneously and on the same surface. This process is, in principle, applicable to any in situ microarray synthesis method, for instance, inkjet or electrochemical DNA synthesis. Full article

DNA data storage based on synthetic oligonucleotides is a major attraction due to the possibility of storage over long periods. Nowadays, the quantity of data generated has been growing exponentially, and the storage capacity needs to keep pace with the growth caused by new technologies and globalization. Since DNA can hold a large amount of information with a high density and remains stable for hundreds of years, this technology offers a solution for current long-term data centers by reducing energy consumption and physical storage space. Currently, research institutes, technology companies, and universities are making significant efforts to meet the growing need for data storage. DNA data storage is a promising field, especially with the advancement of sequencing techniques and equipment, which now make it possible to read genomes (i.e., to retrieve the information) and process this data easily. To overcome the challenges associated with developing new technologies for DNA data storage, a message encoding and decoding exercise was conducted at a Brazilian research center. The exercise performed consisted of synthesizing oligonucleotides by the phosphoramidite route. An encoded message, using a coding scheme that adheres to DNA sequence constraints, was synthesized. After synthesis, the oligonucleotide was sequenced and decoded, and the information was fully recovered. Full article

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5′-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide. Full article

A formaldehyde-free reactive flame retardant, an ammonium salt of triethylenetetramine phosphoryl dimethyl ester phosphamide phosphoric acid (ATPEPDPA), was synthesized and characterized using nuclear magnetic resonance (NMR). Fourier transform infrared spectroscopy test (FT-IR), durability test and scanning electron microscopy (SEM) results suggested that ATPEPDPA was successfully grafted on cotton fabrics through a -N-P(=O)-O-C covalent bond. Moreover, the limiting oxygen index (LOI) value of 20 wt% ATPEPDPA-treated cotton was 44.6%, which met stringent washing standard after 50 laundering cycles (LCs). The high washing resistance of the ATPEPDPA-treated cotton was due to the p-π conjugation between the N atom and the P(=O) group in the flame-retardant molecule, which strengthened the stability of the -N-P(=O)-O-C bonds between ATPEPDPA and cellulose, and the -N-P(=O)-(O-CH₃)₂ groups in the ATPEPDPA. The cone calorimetric test showed that the treated cotton had excellent flame retardance. In addition, the TG and TG-IR tests suggested that ATPEPDPA performed a condensed flame retardance mechanism. Furthermore, the physical properties and hand feel of the treated cotton were well maintained. These results suggested that introducing -N-P(=O)-(O-CH₃)₂ and -N-P(=O)-(ONH₄)₂ groups into ATPEPDPA could significantly increase the fire resistance and durability of cotton fabrics. Full article

A template-assisted assembly approach to a C₂₄ fullerene-like double-stranded DNA polyhedral shell is proposed. The assembly employed a supramolecular oligonucleotide dendrimer as a 3D template that was obtained via the hybridization of siRNA strands and a single-stranded DNA oligonucleotide joined to three- or four-way branched junctions. A four-way branched oligonucleotide building block (a starlet) was designed for the assembly of the shell composed of three identical self-complementary DNA single strands and a single RNA strand for hybridization to the DNA oligonucleotides of the template. To prevent premature auto-hybridization of the self-complementary oligonucleotides in the starlet, a photolabile protecting group was introduced via the N³-substituted thymidine phosphoramidite. Cleavable linkers such as a disulfide linkage, RNase A sensitive triribonucleotides, and di- and trideoxynucleotides were incorporated into the starlet and template at specific points to guide the post-assembly disconnection of the shell from the template, and enzymatic disassembly of the template and the shell in biological media. At the same time, siRNA strands were modified with 2′-OMe ribonucleotides and phosphorothioate groups in certain positions to stabilize toward enzymatic digestion. We report herein a solid-phase synthesis of branched oligodeoxy and oligoribonucleotide building blocks for the DNA/RNA dendritic template and the branched DNA starlet for a template-assisted construction of a C₂₄ fullerene-like DNA shell after initial molecular modeling, followed by the assembly of the shell around the DNA-coated RNA dendritic template, and visualization of the resulting nanostructure by transmission electron microscopy. Full article

Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a neutral phosphorodiamidate linkage. PMOs work via a steric blocking mechanism and are stable towards nucleases’ inside cells. PMOs are usually synthesized using phosphoramidate P(V) chemistry. In this review, we will discuss the synthesis of PMOs, phosphoroamidate morpholinos (MO), and thiophosphoramidate morpholinos (TMO). Full article

Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5′ or 3′ end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation. Full article