Amino-modified Oligonucleotides Research Articles

Front Cover: Unique Crosslinking Properties of Psoralen‐Conjugated Oligonucleotides Developed by Novel Psoralen N‐Hydroxysuccinimide Esters (ChemBioChem 15/2023)

Psoralens and their derivatives have unique crosslinking features to DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable. In this study, we developed novel psoralen N-hydroxysuccinimide esters that allow the introduction of psoralens into any amino-modified oligonucleotides. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools. More information can be found in the Research Article by A. Yamayoshi et al.

Unique Crosslinking Properties of Psoralen-Conjugated Oligonucleotides Developed by Novel Psoralen N-Hydroxysuccinimide Esters.

Psoralens and their derivatives, such as trioxsalen, have unique crosslinking features to DNA. However, psoralen monomers do not have sequence-specific crosslinking ability with the target DNA. With the development of psoralen-conjugated oligonucleotides (Ps-Oligos), sequence-specific crosslinking with target DNA has become achievable, thereby expanding the application of psoralen-conjugated molecules in gene transcription inhibition, gene knockout, and targeted recombination by genome editing. In this study, we developed two novel psoralen N-hydroxysuccinimide (NHS) esters that allow the introduction of psoralens into any amino-modified oligonucleotides. Quantitative evaluation of the photo-crosslinking efficiencies of the Ps-Oligos to target single-stranded DNAs revealed that the crosslinking selectivity to 5-mC is the unique feature of trioxsalen. We found that the introduction of an oligonucleotide via a linker at the C-5 position of psoralen can promote favorable crosslinking to target double-stranded DNA. We believe our findings are essential information for the development of Ps-Oligos as novel gene regulation tools.

A facile and high-sensitive bio-sensing of the V617F mutation in JAK2 gene by GSH-CdTe-QDs FRET-based sensor

This study aimed to directly detect the V617F point mutation of the Janus kinase 2 (JAK2) gene in the target DNA using a FRET-based biosensor. The water-soluble GSH-CdTe-QDs were synthesized by a one-step process, then GSH-QD conjugated to the termini amino-modified oligonucleotides target via carboxylic groups on the QD surface. The prepared QDs-DNA biosensor was applied in the quantitative and rapid detection of V617F mutation with a detection limit of 3 × 10−9 mol L−1 based on the FRET mechanism. In other words, detecting the V617F mutation by bio-sensing technology would be much simpler, cheaper, time-saving, highly sensitive, and more convenient than molecular diagnostic tools. Furthermore, the nano-biosensor was applied to detect the V617F mutation in clinical samples compared to the common ARMS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) standard method.The results revealed that the GSH-capped biosensors would be effective for V617F mutation detection in samples distinguished with satisfactory analytical outcomes. Therefore, the designed fluorescence nanoprobe is suitable for the specific detection of V617F mutation of the JAK2 gene in clinical samples.

Direct ultrasensitive electrochemical detection of breast cancer biomarker-miRNA-21 employing an aptasensor based on a microgel nanoparticle composite

Breast cancer is a common disease with high morbidity and mortality among women worldwide. Serum miRNA-21 expression is higher in breast cancer patients and can be employed as a predictor for early breast cancer diagnosis. There is an urgent need for a sensitive, convenient and cost-effective biosensor to assist in the direct diagnosis of breast cancer through clinical biomarkers. In this article, a highly sensitive microgel nanocomposite-based electrochemical aptasensor is prepared for the detection of miRNA-21. The microgel particles of gold nanoparticles are wrapped in a mixture of acrylic acid (AAc) and N-isopropylacrylamide (NIPAm) polymerization. This leads to a porous network structure which can provide a highly biocompatible and anti-fouling microenvironment, similar to that of aqueous biological tissue for RNA capture reaction. Complementary to miRNA-21, an amino-modified oligonucleotide chain capture probe is covalently immobilized on the microgel material, while the hybridization of miRNA-21 with the capture probe is monitored by different pulse voltammetry (DPV). The sensing platform can directly and accurately detect target miRNAs in serum without sample preparation and purification. The concentration response of the aptasensor presents a linear range of 10 aM to 1 pM as well as a detection limit of 1.35 aM. The aptasensor also exhibits noticeable selectivity, stability and reproducibility. Simultaneously, we successfully apply this method to the subsequent detection of serum miRNA-21 in 2 breast cancer patients before treatment and after recovery respectively. The consequence proves that the method has promising potential applications in the early diagnosis and treatment of breast cancer patients.

Light-Induced Oxidase Activity of DNAzyme-Modified Quantum Dots.

Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H2O2-free DNAzyme system with oxidase activity. Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH2) and carboxylated quantum dots (CdTe@COOH QDs). The obtained products were characterized by spectroscopic methods (UV-Vis, fluorescence, circular dichroizm (CD), and IR) and the transmission electron microscopy (TEM) technique. A QD-DNA system with a low polydispersity and high stability in aqueous solutions was successfully obtained. The catalytic activity of the QD-DNA conjugate was examined with Amplex Red and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) indicators using reactive oxygen species (ROS) generated by visible light irradiation. The synthesized QD-DNAzyme exhibited enhanced catalytic activity compared with the reference system (a mixture of QDs and DNAzyme). This proved the assumption that the covalent attachment of DNAzyme to the surface of QD resulted in a beneficial effect on its catalytic activity. The results proved that the QD-DNAzyme system can be used for generation of the signal by light irradiation. The light-induced oxidase activity of the conjugate was demonstrated, proving that the QD-DNAzyme system can be useful for the development of new cellular bioassays, e.g., for the determination of oxygen radical scavengers.

Conjugation of Oligonucleotides to Peptide Aldehydes via a pH-Responsive N-Methoxyoxazolidine Linker.

The formation of N-methoxyoxazolidines in the preparation of oligonucleotide-peptide conjugates was evaluated. The reaction occurred between unprotected 2'-N-(methoxy)amino-modified oligonucleotides and peptide aldehydes in reasonable yields when isolated. The reaction is reversible under slightly acidic conditions, and it is pH-responsive. The rate and the equilibrium constant may be varied with structurally different aldehydes, allowing an optimization of the ligation and cleavage rate of the resultant conjugates. Therefore, this concept can be considered a cleavable linker.

Pegylation of RNA Spiegelmers by a Novel Widely Applicable Two-Step Process for the Conjugation of Carboxylic Acids to Amino-Modified Oligonucleotides.

The reaction between N-hydroxy succinimide (NHS) ester-activated carboxylic acids and amino-modified nucleic acids is commonly used for the post-synthetic modification of oligonucleotides. Here, we report a two-step variation of the method in which the NHS ester is replaced by the corresponding parent carboxylic acid. In the first step, the carboxylic acid is activated with a standard peptide coupling reagent like HBTU in an anhydrous water-miscible aprotic organic solvent. In the second step, the solution of the activated carboxylic acid is added to the amino-modified oligonucleotide in water. The method is demonstrated using 40-kDa polyethylene glycol (PEG) carboxylic acid and biotin as examples. Recycling of the carboxylic acid, which is typically used in molar excess over the nucleic acid, is shown for the conjugation with 40-kDa PEG carboxylic acid. This conjugation method is generally applicable to the conjugation of carboxylic acids to amino-modified oligonucleotides, thus enabling the attachment of small to large molecular entities such as dyes, tags, peptides, and other macromolecules. © 2020 Wiley Periodicals LLC. Basic Protocol 1: General protocol for the conjugation of an amino-modified oligonucleotide with a carboxylic acid, exemplified for 40-kDa PEG carboxylic acid Basic Protocol 2: Biotinylation of an amino-modified oligonucleotide using the general conjugation protocol Basic Protocol 3: Recycling of the carboxylic acid component from the conjugation reaction, demonstrated for 40-kDa PEG carboxylic acid using ultrafiltration Support Protocol 1: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with 40-kDa PEG carboxylic acid Support Protocol 2: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with biotin Support Protocol 3: Analytical IP-RP-HPLC method used as in-process control method to monitor the conjugation reaction Alternate Protocol: Separation of 40-kDa PEG carboxylic acid from unreacted and conjugated oligonucleotide by preparative AEX-HPLC.

An Electrochemiluminescence Sensor Based on CdSe@CdS and God Signal Amplification for Sensitive Detection of Hg2+

Mercury is highly toxic, so it is important to develop a rapid and sensitive method for the detection of mercury. In this paper, mercaptopropionic acid-capped core-shell CdSe@CdS quantum dots (QDs) were synthesized by one-step hydrothermal method[1]. Molybdenum disulfide (MoS2) nanosheets were prepared by the method which we previously reported[2]. 5'-amino-modified oligonucleotide capture probe DNA1 (5'-NH2-ss-DNA) and 5'-thiol-modified oligonucleotide signal probe DNA2 (5'-SH-ss-DNA) was synthesized by Shanghai Bioengineering Technology Co., Ltd. (China). The sequence (5' to 3') is as follows[3]: DNA1：5'-NH2-AAAATTTTGCTTTGGTTT-3' DNA2：5'-SH-AAAAATTTCCTTTGCTTTT-3' Polycationic poly(diallyldimethylammonium chloride) (PDDA) and negatively charged QDs were adsorbed on the surface of MoS2, in sequence, to form the QDs- PDDA-MoS2 composites, which were employed as a matrix for the immobilization of DNA1 strand[1]. Gold nanoparticles (AuNPs) were prepared and were combined with the 5'-thiol-modified DNA2 probe and glucose oxidase (GOD) to prepare GOD-AuNPs-DNA2 conjugates[2]. First, the QDs-PDDA-MoS2 nanocomposites were modified onto the surface of a glassy carbon electrode (GCE) as a carrier for preparing an electrochemiluminescence (ECL) sensor. The QDs-PDDA-MoS2/GCE electrode was immersed in an aqueous solution consists of 10.0 mM ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and 5.0 mM N-hydroxysuccinimide (NHS) for 30 min to activate the terminal carboxylic acid group of QDs. DNA1 could immobilize onto the surface of the CdSe@CdS QDs via the interaction between the activated carboxylic acid group and the amino group of DNA1. The modified electrode was denoted as DNA1/QDs-PDDA-MoS2/GCE. The schematic diagram of fabrication of the sensor was shown in Fig. 1. Fig. 1 Frabrication process and the detection machanisms of the ECL sensor. Insert: curve (a) absence and (b) presence of Hg2+. Electrochemical measurement for ECL was carried out on an MPI-A detection system (Xi'an Rimax Electronics Co., Ltd. China) with a three-electrode system and a photomultiplier tube. The voltage was set at 800 V. A glassy carbon electrode (diameter, 3 mm) was used as a working electrode; a platinum wire and an Ag/AgCl (saturated KCl solution) were used as a counter electrode and a reference electrode, respectively. The DNA1/QDs-PDDA-MoS2/GCE electrodes were incubated with GOD-AuNPs-DNA2 suspension and different concentrations of Hg2+ solution in turns. Then the electrodes were immersed in 0.1 M phosphate buffer solution containing 1% glucose to measure ECL signal, respectively. The potential window was from 0 to 1.5 V with a scan rate of 100 mV/ s. Hg2+ ions can specifically bind to the T-T mismatch base of DNA1 and DNA2 to form the T-Hg2+-T complex. Therefore, GOD-AuNPs-DNA2 can be modified to the electrode surface. GOD can catalyze the oxidation reaction of dissolved oxygen molecules towards glucose in the solution to produce H2O2 which is a co-reactant and can enhance the ECL of the QDs. The ECL signal increased linearly with Hg2+ concentration from 1.0×10-12 to 1.0×10-6 M. The detection limit of 0.1 pM can be estimated at a signal-to-noise ratio of 3. The ECL sensor has the advantages of simple preparation method, high sensitivity, good specificity and wide linear range for the determination of Hg2+ ions.

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester.

We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0–8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39–85% yield. This method has potential uses in biotechnology and chemical biology.

Optimized Reaction Conditions for Amide Bond Formation in DNA-Encoded Combinatorial Libraries.

DNA-encoded combinatorial libraries are increasingly being used as tools for the discovery of small organic binding molecules to proteins of biological or pharmaceutical interest. In the majority of cases, synthetic procedures for the formation of DNA-encoded combinatorial libraries incorporate at least one step of amide bond formation between amino-modified DNA and a carboxylic acid. We investigated reaction conditions and established a methodology by using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, 1-hydroxy-7-azabenzotriazole and N,N'-diisopropylethylamine (EDC/HOAt/DIPEA) in combination, which provided conversions greater than 75% for 423/543 (78%) of the carboxylic acids tested. These reaction conditions were efficient with a variety of primary and secondary amines, as well as with various types of amino-modified oligonucleotides. The reaction conditions, which also worked efficiently over a broad range of DNA concentrations and reaction scales, should facilitate the synthesis of novel DNA-encoded combinatorial libraries.

Quantum dot-DNA aptamer conjugates coupled with capillary electrophoresis: A universal strategy for ratiometric detection of organophosphorus pesticides

Based on the highly sensitivity and stable-fluorescence of water-soluble CdTe/CdS core–shell quantum dots (QDs) with broad-specificity DNA aptamers, a novel ratiometric detection strategy was proposed for the sensitive detection of organophosphorus pesticides by capillary electrophoresis with laser-induced fluorescence (CE–LIF). The as-prepared QDs were first conjugated with the amino-modified oligonucleotide (AMO) by amidation reaction, which is partial complementary to the DNA aptamer of organophosphorus pesticides. Then QD-labeled AMO (QD-AMO) was incubated with the DNA aptamer to form QD-AMO–aptamer duplex. When the target organophosphorus pesticides were added, they could specifically bind the DNA aptamer, leading to the cleavage of QD-AMO–aptamer duplex, accompany with the release of QD-AMO. As a result, the ratio of peak height between QD-AMO and QD-AMO–aptamer duplex changed in the detection process of CE–LIF. This strategy was subsequently applied for the detection of phorate, profenofos, isocarbophos, and omethoate with the detection limits of 0.20, 0.10, 0.17, and 0.23μM, respectively. This is the first report about using QDs as the signal indicators for organophosphorus pesticides detection based on broad-specificity DNA aptamers by CE–LIF, thus contributing to extend the scope of application of QDs in different fields. The proposed method has great potential to be a universal strategy for rapid detection of aptamer–specific small molecule targets by simply changing the types of aptamer sequences.

Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide

The functionalization of silicon oxide based substrates using silanes is generally performed through liquid phase methodologies. These processes involve a huge quantity of potentially toxic solvents and present some important disadvantages for the functionalization of microdevices or porous materials, for example the low diffusion. To overcome this drawback, solvent-free methodologies like molecular vapor deposition (MVD) or supercritical fluid deposition (SFD) have been developed. In this paper, the deposition process of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon oxide using supercritical carbon dioxide (scCO2) as a solvent is studied for the first time. The oxirane ring of epoxy silanes readily reacts with amine group and is of particular interest for the grafting of amino-modified oligonucleotides or antibodies for diagnostic application. Then the ability of this specific EBTMOS layer to react with amine functions has been evaluated using the immobilization of amino-modified oligonucleotide probes. The presence of the probes is revealed by fluorescence using hybridization with a fluorescent target oligonucleotide. The performances of SFD of EBTMOS have been optimized and then compared with the dip coating and molecular vapor deposition methods, evidencing a better grafting efficiency and homogeneity, a lower reaction time in addition to the eco-friendly properties of the supercritical carbon dioxide. The epoxysilane layers have been characterized by surface enhanced ellipsometric contrast optical technique, atomic force microscopy, multiple internal reflection infrared spectroscopy and X-ray photoelectron spectroscopy. The shelf life of the 3,4-epoxybutyltrimethoxysilane coating layer has also been studied. Finally, two different strategies of NH2-oligonucleotide grafting on EBTMOS coating layer have been compared, i.e. reductive amination and nucleophilic substitution, SN2. This EBTMOS based coating layer can be used for a wide range of applications such as the preparation of new supported and recoverable catalysts and new integrated silicon microdevices for healthcare purposes.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor.

We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.

Visual DNA microarray for detection of epidermal growth factor receptor (EGFR) gene mutations

Objective. To develop a gold nanoparticle-based visual DNA microarray for simple and rapid screening of EGFR gene mutations. Methods. The DNA fragments contain epidermal growth factor receptor (EGFR) exons 18, 19, 20 and 21 were amplified by polymerase chain reaction (PCR) using biotin-modified primers. The amino-modified oligonucleotides were immobilized on glass surface, which were used as the capturing probes to bind the complement biotinylated target DNA. After the PCR product has been hybridized to the immobilized capture probe DNA on the glass slides, the Streptavidin-conjugated gold nanoparticles were introduced to the microarray via specific binding to 5ʹ-end biotin of the PCR products. The hybridization signal on array spots was enhanced and visualized by silver amplification. The EGFR mutation in 286 clinical samples from cancer patients were tested using the gold nanoparticle-based microarray and verified with Sanger DNA sequencing method. Results. A novel visual DNA microarray has been developed to detect EGFR mutations in tumor tissue specimens rapidly; its limit of detection (LOD) is up to 10−9 mol/L and distinguishes power to detect 5% gene mutation in the mixture samples. Conclusion. For its high specificity and sensitivity, simplicity, lower price and higher speed, the present visual mutation detecting technique has potential application in clinical fields.

Synthesis and Application of Highly Reactive Amino Linkers for Functional Oligonucleotides

Oligonucleotides are functionalized by conjugation with a variety of molecules, and aliphatic amino linkers have been frequently used as a tether for their modifications. This unit describes the syntheses and applications of novel amino linkers having a carbamate structure. Two major chemical properties of the primary amine are induced by the neighboring effect of the carbamate group, which are found to be optimum in an aminoethyl carbamate structure. First, the hydrophobic monomethoxytrityl group can be rapidly removed from the aminoethyl carbamate under very mild acidic conditions, while the deprotection is not completed in standard aliphatic amines even under high acid concentration. This significant feature enables the convenient purification of amino-modified oligonucleotides by using the hydrophobic interaction of the monomethoxytrityl group with a reverse-phase resin. Second, the introduction of the carbamate linkage reduces the pK(a) value of the neighboring primary amine, resulting in an increase in the conjugation yields with various functional molecules, such as those having active esters. The novel amino linkers that have an aminoethyl carbamate linkage indicate potent activity and are applicable for the preparation of various functional oligonucleotides.

Synthesis of SERS active nanoparticles for detection of biomolecules

Surface Enhanced Raman Scattering (SERS) can be used to detect specific DNA sequences by methods based on hybridisation of oligonucleotide functionalized nanoparticles to complementary DNA sequences. The problem, which has to be overcome to use this technique is that DNA is not strongly SERS active. This is due to the lack of a visible chromophore and presence of the highly negatively charged phosphate backbone, which prevents the electrostatic interaction with the metal surface necessary for the enhancement. To obtain SERS active DNA a label containing a surface seeking group, to allow adsorption of DNA on a metal surface, and a chromophore has to be attached to the DNA strand. Here we report the synthesis of three linkers containing a Raman tag [the following fluorophores were used for this purpose due to the fluorescence quenching ability of metallic nanoparticles: fluorescein, 6-aminofluorescein and tetramethylrhodamine (TAMRA)], surface complexing group (cyclic disulphide—thioctic acid) and a chemical functionality for attachment of DNA (carboxyl group). Each of the linkers also contain poly(ethylene glycol) (PEG) (3 mer), which reduces non-specific adsorption of molecules to the surface of the nanoparticles and provides colloidal stability. The synthesized linkers were used to functionalize gold citrate (18 and 50 nm), silver citrate (40 nm) and silver EDTA (35 nm) nanoparticles. All of the conjugates exhibit high stability, gave good SERS responses at laser excitation frequencies of 514 and 633 nm and could be conjugated to amino-modified oligonucleotides in the presence of the commonly used ( N-(3-dimethylaminopropyl)- N′-ethyl carbodiimide hydrochloride—EDC·HCl with N-hydroxysulfosuccinimide or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride—DMT MM, which has not been used for bioconjugate preparation previously. This approach is less time consuming and less expensive than previously used protocols and does not require the formation of a mixed layer of oligonucleotides and Raman reporter on the metal surface. Additionally the presence of a reactive functionality within the linker structure makes it possible to conjugate the linker to other biomolecules of interest such as proteins.

Oxygen Plasma Treated Interactive Polycarbonate DNA Microarraying Platform

A novel DNA microarrying platform based on oxygen plasma activation of polycarbonate surface of compact disks (DVD) is presented. Carboxylic acid groups are generated in few seconds on polycarbonate in an efficient, fast, and clean way. Following this surface activation strategy, amino-modified oligonucleotide probes were covalently attached, reaching an immobilization density of 2 pmol cm(-2). Atomic force microscopy imaging revealed the nondestructive character of this treatment when applied for short times, allowing for disk scanning in standard DVD drives. DNA assays performed on oxygen plasma treated disks resulted very efficient with maximum hybridization yield of 93% and reaching a low limit of detection (200 pM) for perfect match synthetic oligonucleotide targets when reading the disk with a standard drive as detector. The approach was also evaluated by scoring single nucleotide polymorphisms with a discrimination ratio of 12.8. As proof of concept, the oxygen plasma treated interactive polycarbonate DNA microarraying platform was applied to the detection of PCR products of Salmonella spp., reaching a detection limit of 2 nM that corresponds to a DNA concentration of only 1 c.f.u./mL. The results confirm the suitability of the microarray platform for analysis of biological samples with high sensitivity.

RNA Aptamers and Spiegelmers: Synthesis, Purification, and Post‐Synthetic PEG Conjugation

This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the L-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.

Detection and comparison of protein–DNA interactions using DNA–BIND plate and horseradish peroxidase-based colorimetric assay

We describe a procedure for detection and comparison of protein–DNA interactions using DNA–BIND plate and horseradish peroxidase (HRP)-based colorimetric assay. Amino-modified oligonucleotide was covalently immobilized on the surface of DNA–BIND plate. After the complementary oligonucleotide was annealed, the plate was incubated with protein to allow sequence-specific DNA binding. Primary antibody and HRP-labeled secondary antibody were then employed, and colorimetric assay was conducted before the absorbance was read. This is a sensitive, specific, and high-throughput method that has been applied not only in the detection of protein–DNA interaction but also in the quantitative comparison of DNA-binding capabilities among wild-type and mutant proteins.

Impedimetric genosensing of DNA polymorphism correlated to cystic fibrosis: A comparison among different protocols and electrode surfaces

In this work, a genosensor for the impedimetric detection of the triple base deletion in a cystic fibrosis-related DNA synthetic sequence is presented. Screen-Printed Carbon Electrodes containing Carboxyl functionalised multi-walled carbon nanotubes were used for the immobilization of an amino-modified oligonucleotide probe, complementary to the Cystic Fibrosis (CF) mutant gene. The complementary target (the mutant sequence) was then added and its hybridization allowed. The change of interfacial charge transfer resistance ( R ct) between the solution and the electrode surface, experimented by the redox marker ferrocyanide/ferricyanide, confirmed the hybrid formation. A non-complementary DNA sequence and a three-mismatch sequence corresponding to the wild DNA gene (present in healthy people) were used as negative controls. A further step employing a signalling biotinylated probe was performed for signal amplification using streptavidin-modified gold nanoparticles (strept-AuNPs). In order to observe by SEM the presence and distribution of strept-AuNPs, a silver enhancement treatment was applied to electrodes already modified with DNA–nanoparticles conjugate. The developed protocol allowed the very sensitive detection of the triple base deletion in a label-free CF-related DNA sequence, achieving a LOD around 100 pM. Results were finally compared with those obtained using different protocols for immobilization of DNA capture probe.