Short-chain Oligonucleotides Research Articles

POSoligo software for in vitro gene synthesis

Oligonucleotide synthesis is vital for molecular experiments. Bioinformatics has been employed to create various algorithmic tools for the in vitro synthesis of nucleotides. The main approach to synthesizing long-chain DNA molecules involves linking short-chain oligonucleotides through ligase chain reaction (LCR) and polymerase chain reaction (PCR). Short-chain DNA molecules have low mutation rates, while LCR requires complementary interfaces at both ends of the two nucleic acid molecules or may alter the conformation of the nucleotide chain, leading to termination of amplification. Therefore, molecular melting temperature, length, and specificity must be considered during experimental design. POSoligo is a specialized offline tool for nucleotide fragment synthesis. It optimizes the oligonucleotide length and specificity based on input single-stranded DNA, producing multiple contiguous long strands (COS) and short patch strands (POS) with complementary ends. This process ensures free 5′- and 3′-ends during oligonucleotide synthesis, preventing secondary structure formation and ensuring specific binding between COS and POS without relying on stabilizing the complementary strands based on Tm values. POSoligo was used to synthesize the linear RBD sequence of SARS-CoV-2 using only one DNA strand, several POSs for LCR ligation, and two pairs of primers for PCR amplification in a time- and cost-effective manner.

Attomolar-level detection of respiratory virus long-chain oligonucleotides based on FRET biosensor with upconversion nanoparticles and Au–Au dimer

Upconversion nanoparticles (UCNPs) are promising nanoprobes in DNA/RNA detection, such as respiratory viral RNAs, and siRNA in cancer. However, recent studies have indicated that the sensitivity of UCNP-based biosensors is restricted, ranging from picomolar to femtomolar level. Moreover, most of existing UCNP-based probes are only able to detect short-chain oligonucleotides, which are not suitable for detection of long-chain oligonucleotides in many real applications. In this work, we introduced a new UCNP-based fluorescence resonance energy transfer (FRET) nanoprobe design composed of NaGdF4:Yb3+, Er3+@NaGdF4 core-shell UCNPs (csUCNPs) linking with Au–Au dimer to detect long-chain oligonucleotides of SARS-CoV-2 N-gene. Compared with typical single gold nanoparticles (AuNPs) in FRET biosensors, our theoretical investigation shows that a stronger electromagnetic field is generated in the Au–Au dimer where the plasmon resonance can enhance FRET efficiency and increase the working distance. Thus, the synergetic effect of plasmonic resonance and FRET enables a greater quenching efficiency (QE) of Au–Au dimer to UCNPs, which leads to more remarkable upconversion luminescence (UCL) recovery for each target gene recognition. Importantly, our design significantly improved the limit of detection (LOD) to attomolar level, with a linear response ranging from 2 aM to 2 fM. Moreover, the clinical detection with inactivated SARS-CoV-2 samples was successfully performed with excellent specificity within 30 min using the developed UCNPs biosensors incorporated with Au–Au dimer. This UCNP biosensor based on Au–Au dimer strategy with ultra-sensitivity and good selectivity opens a new path for clinical diagnosis without target amplification and plays an instructive role in other virus diagnosis.

Therapeutic impact of Telomerase Inhibitor Imetelstat: A Literature Review

The immortal phenotype of cancer cells is a significant trait, and in the vast majority of malignancies, the enzyme telomerase is essential for sustaining the cancer cells' limitless capacity for replication. The absence of telomerase expression and the lack of an immortal phenotype in normal adult tissues suggest that telomerase is a prospective therapeutic target for the treatment of a variety of tumor forms. At every cell division, telomeres will shrink if telomerase is not active. Apoptosis, cell senescence, and chromosomal instability are initiated when the telomeres become critically short. Telomeres are stabilized in the majority of rapidly expanding cancers by reactivating telomerase. In numerous tumor forms, it has been demonstrated that telomerase inhibition inhibits the development of cancer cells. Telomerase inhibitors have been discovered over the past 10 years as a result of substantial basic research into the mechanisms regulating telomeres, which may offer a potent, nearly universal cancer treatment approach. A short-chain oligonucleotide called imetelstat [GRN163L, Geron Corporation] has a high affinity and specificity for the RNA component of telomerase's template region [hTR or hTERC]. Researchers conduct an examination of articles that are in accordance with the issue to be studied. Articles used in literature review are obtained through the database of international journal providers through PubMed, we investigated clinical studies and discussed what happened in these clinical studies and the extent of the effectiveness of imetelstat in treatment of cancer. Articles proved that imetelstat could enhance cancer treatment. Articles proved that Imetelstat is promising therapeutic agents for cancer treatment. In this review, we suggest that formulating and following treatment, Further studies are needed to determine the related mechanisms to enhance Imetelstat efficacy.

Investigation of the Influence of Charge State and Collision Energy on Oligonucleotide Fragmentation by Tandem Mass Spectrometry.

Due to the increasing pharmaceutical interest of oligonucleotides, for example in antisense therapy and vaccines, their analytical characterization is of fundamental importance due to their complex structure. For this purpose, mass spectrometry is a viable tool for structural studies of nucleic acids. Structural information regarding the primary sequence of a nucleic acid can reliably be gained via tandem mass spectrometry (MSMS) fragmentation. In this work, we present the characteristic fragmentation behavior of short-chain oligonucleotides (15-35 nucleotides) with respect to the collision-induced dissociation (CID) voltage used. The relationship and influence of the length of the oligonucleotide and its charge state is also discussed. The results presented here can be helpful for estimating the required fragmentation energies of short-chain oligonucleotides and their sequencing.

Isolation of DNA Aptamers for Enteropathogenic Escherichia coli (EPEC) Detection using Bacterial-SELEX Approach

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative pathogenic bacterium that causes diarrheal disease, especially in infants and children. Aptamers are short chain oligonucleotides that have high affinity, specificity, and selectivity to their targets, which have potential to be developed as a method for diagnosing pathogens. In this study, aptamer was isolated through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method using whole cells bacteria (Bacterial-SELEX) for recognizing pathogenic E. coli EPEC K1.1 which was isolated from children with diarrhea in Indonesia. Ten rounds of bacterial-SELEX procedure were conducted with modification conditions by using Top10, DH5a E. coli cells, Listeria monocytogenes, and Lactobacillus plantarum S34 as counter-selections. The selection process was started with a pool of ssDNA random library consisting of a random base with 40-nucleotides long flanked with fixed primers sequence for aptamer amplification purpose. Short single-stranded DNA amplification was done by symmetric and asymmetric PCR. The highly enriched oligonucleotide pools (pooled 8, 9, and 10) were cloned and the resulting ssDNA aptamers were identified by Sanger DNA sequencing. Finally, twelve aptamers with unique sequences and various secondary structures including G-quadruplex sequence motif within aptamers were obtained as candidates specific aptamer for detection and capturing of EPEC K1.1.

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

Aptamer based tools for environmental and therapeutic monitoring: A review of developments, applications, future perspectives

Nucleic acids in the form of aptamers play a growing and significant role in the targeted and rapid analysis of environmental sample composition and medical analyses. In this paper, the review of both aptamers synthesis methods as well as application of these short chain oligonucleotides (with critical comments on their strong and weak features) are given. The first ones include: systematic evolution of ligands by exponential enrichment (SELEX), high throughput aptamer identification screen (HAPIscreen), and a non-equilibrium capillary electrophoresis of equilibrium mixture (NECEEM). Afterwards, manuscript describes variety of sensors and biotests utilizing aptamers as active part of its action starting from electrochemical aptasensors, through optical to piezo-electric ones. Described biotests present basic developments in enzyme-linked apta-sorbent assays (ELASA) that can be performed with different variations (enzyme-linked aptamer assay (ELAA), enzyme-linked oligonucleotide assay (ELONA) and aptamer-linked immobilized sorbent assay (ALISA)). Next, the review presents advantages and drawbacks of recent aptameric developments in versatile laboratory applications, namely medical ones, as well as analytical and bioassays. Utilitarian development of aptasensors and aptamers would strongly benefit from an assembly of interdisciplinary teams containing chemists, physicists, biologists, medical doctors, and material and electronic scientists, to determine the most effective application methodologies.

A Simple and Efficient Microfluidic System for Reverse Chemical Synthesis (5′-3′) of a Short-Chain Oligonucleotide Without Inert Atmosphere

Reverse DNA synthesis (5′-3′) plays diverse functional roles in cellular biology, biotechnology, and nanotechnology. However, current microfluidic systems for synthesizing single-stranded DNAs at a laboratory scale are limited. In this work, we develop a simple and efficient polydimethylsiloxane- (PDMS-) based microfluidic system for the reverse chemical synthesis of short-chain oligonucleotides (in the 5′-3′ direction) under ambient conditions. The use of a microfluidics device and anhydrous conditions effectively surpass the problem of moisture sensitivity during oligonucleotide synthesis. With optimized microfluidic synthesis conditions, the system is able to synthesize up to 21 bases-long oligonucleotides in air atmosphere. The as-synthesized oligonucleotides, without further purification, are characterized using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF/TOF) mass spectroscopy (MS) supported by the denatured polyacrylamide gel electrophoresis (PAGE) analysis. This developed system is highly promising for producing the desired sequence at the nanomolar scale on-chip and on-demand in the near future.

Short-chain oligonucleotide detection by glass nanopore using targeting-induced DNA tetrahedron deformation as signal amplifier

Glass capillary nanopore has been developed as a promising sensing platform for bioassay with single-molecule resolution. Although the diameter of glass capillary nanopore can be easily tuned, direct event-readouts of small biomacromolecules, like short-chain oligonucleotide fragments (within ∼20 nucleotides) remain great challenge, which limited by the configuration of the conical-shaped nanopore and the instrumental temporal resolution. Here, we exploit a smart strategy for glass nanopore detection of short-chain oligonucleotides by using relatively big-sized tetrahedral DNA nanostructures as a signal amplifier, which can amplify the signals and retard the translocation speed meanwhile. The tetrahedral DNA nanostructure with a hairpin loop sequence in one edge, undergoes a shape transformation upon the complementary combination of the target oligonucleotides, in which the presence of short-chain target oligonucleotide can be readout due to obvious variation in amplitude of ion current pulse that caused by volume change of the DNA tetrahedral. Therefore, this strategy is promising for extending glass nanopore sensing platform for sensitive detection of short-chain oligonucleotides.

Prediction, docking study and molecular simulation of 3D DNA aptamers to their targets of endocrine disrupting chemicals

Typical endocrine disrupting chemicals, including BPA (Bisphenol A), E2 (17-β-Estradiol) and PCB 72 (polychlorinated biphenyl 72), are commonly and widely present in the environment with good chemical stability that are difficult to decompose in vitro and in vivo. Most of the high-qualified antibodies are required as the key biomaterials to fabricate the immunosensor for capturing and detecting. As an ideal alternative, the short-chain oligonucleotides (aptamer) are essentially and effectively employed with the advantages of small size, chemical stability and high effectiveness for monitoring these environmental contaminants. However, the molecular interaction, acting site and mode are still not well understood. In this work, we explored the binding features of the aptamers with their targeting ligands. The molecular dynamics simulations were performed on the aptamer–ligand complex systems. The stability of each simulation system was evaluated based on its root-mean-square deviation. The affinities of these proposed ligands and the predicted binding sites are analyzed. According to the binding energy analysis, the affinities between ligands and aptamers and the stability of the systems are BPA > PCB 72 >E2. Trajectory analysis for these three complexes indicated that these three ligands were able to steadily bind with aptamers at docking site from 0 to 50 ns and contributed to alteration of conformation of aptamers.

Accelerated digestion of nucleic acids by pepsin from the stomach of chicken

Nucleic acids have become an important nutritional supplement in poultry feed; however, the digestion of nucleic acids in poultry is unclear. The objective of this study was to investigate the digestion of nucleic acids by chicken pepsin in vitro.The extracted pepsinogen from the stomach of the chicken was purified to homogeneity. Upon activation at pH 2.0, chicken pepsinogen was converted to its active form.Nucleic acids, including λ-DNA, salmon sperm DNA and single-strand DNA (ssDNA), can be used as substrates and digested into short-chain oligonucleotides by pepsin.Interestingly, the digestion of the nucleic acids was inhibited when pepsin was treated by alkaline solution (pH 8.0) or pepstatin A. Also, the digestion of the nucleic acids was not affected by the addition of haemoglobin or bovine serum albumin.The results suggested that nucleic acids could be digested by chicken pepsin. Thus pepsin may have a role in digesting nucleic acids in vivo. Nucleic acids added to poultry fed may be digested, starting from the stomach.

The study of the possibility of gold hydrosol stabilization with nonthiolated homooligonucleotides with different lengths and sequences

The stabilization of citrate gold hydrosols with nonthiolated oligonucleotides having different lengths and sequences has been studied, with the oligonucleotides being added both at the stage of hydrosol synthesis (at 37 and 60°C) and after it. The oligonucleotides have been found to exhibit a pronounced stabilizing effect irrespective of the moment of their addition to the hydrosols. The only exception is the situation, in which adenine-based oligonucleotides are heated to 60°C in the course of the synthesis, when they, on the contrary, destabilize the hydrosols. Short-chain oligonucleotides (hexamers) exhibit a stronger stabilization effect than dodecamers do. Moreover, it has been shown that adenine-based and cytosine-based oligonucleotides show the strongest stabilizing effect.

Stimuli-responsive polymers in gene delivery

Recent interest in clinical therapy has been directed to deliver nucleic acids (DNA, RNA or short-chain oligonucleotides) that alter gene expression within a specific cell population, thereby manipulating cellular processes and responses, which in turn stimulate immune responses or tissue regeneration, or blocks expression at the level of transcription or translation for treatment of several diseases. Both ex vivo and in vivo gene delivery can be achieved mostly by using a delivery system (vector). Viral vectors exhibit high gene expression, but also have very significant side effects. Mainly cationic polymeric systems are used as nonviral vectors, although usually with low levels of transfection. Through the use of stimuli-responsive polymers as novel vectors for gene delivery, two benefits can be obtained: high gene expression efficiency and more selective gene expression.

Stabilization of DNA duplexes by covalently-linked peptides

The hybridization properties of various peptide–oligonucleotide hybrids were assessed from UV thermal denaturation experiments. Analysis of these and other published data suggests that peptide chains, both hydrophobic and positively charged, generally have a clear stabilizing effect on short-chain oligonucleotide duplexes.

Stabilization of DNA duplexes by covalently-linked peptides

The hybridization properties of various peptide–oligonucleotide hybrids were assessed from UV thermal denaturation experiments. Analysis of these and other published data suggests that peptide chains, both hydrophobic and positively charged, generally have a clear stabilizing effect on short-chain oligonucleotide duplexes.

The design of antisense RNA.

Antisense nucleic acids comprise short-chain synthetic oligonucleotides, often oligodeoxyribonucleotides (ODN) of less than approximately 30 nucleotides and substantially longer sequences formed by ribonucleic acids (RNA). Both groups differ with respect to several properties, including their generation, the mode of delivery, and their structure-function relationship. Long-chain antisense RNA transcribed in vitro or endogenously from recombinant genes fold into three-dimensional structures. The pairing reaction with their complementary target strand occurs via largely unknown annealing mechanisms and, depending on the phylogenetic cellular background, in different cellular compartments. The annealing pathway is influenced by a variety of biologic and biochemical parameters that are as yet poorly understood. However, the basal biochemical mechanisms underlying the relationship between RNA structure and efficient annealing could allow one to derive more general rules for the design of in vivo effective antisense RNA in a way that is not dependent on specific cell types. Here, some of the criteria are discussed that are currently thought to have major impact on the design of long-chain antisense RNA.

Enhanced resolution in the fingerprinting of short-chain oligonucleotides using the dodecasodium salt of phytic acid in capillary electrophoresis.

The inclusion of the dodecasodium salt of phytic acid in separation buffers has been found to improve considerably the separation of a number of synthetic short-chain oligonucleotides. A combination of the simplicity and the improved resolving power of this methodology allows the facile analysis of mixtures of such compounds. The presence of a large concentration of sodium ions reduces electroosmotic mobility due to more efficient shielding of negative charges on the inner surface of the capillary wall. This, in turn, leads to a more gradual fall in potential between this surface and the center of the capillary.

Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates.

Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of several types of rpc-5-like resins and their use for the separation of oligonucleotides and mononucleotides by high-performance liquid chromatography

Improved alternative packing materials to RPC-5 were prepared from granular polychlorotrifluoroethylene of several different particle sizes and trioctylmethylammonium chloride, and used for the separation of mononucleotides and oligonucleotides in high-performance liquid chromatography. High resolution was obtained when particles of 4–8 μm diameter were used, whereas coarser materials (40–90 or 10–40 μm) gave poor resolution. The greater the amount of the quaternary ammonium salt coated, the smaller was the elution volume for the same sample. Oligonucleotides up to a chain length of 75 could be successively separated in less than 2 h under the optimum conditions. Resolution of mononucleotides and short-chain oligonucleotides was accomplished with the resin coated with a minimum amount of the quaternary ammonium salt. A variety of oligonucleotides could be separated depending on the chain length, the differences in the internucleotide linkage and the differences in the base sequence.

Analysis of conformational parameters in nucleic acid fragments. IV. Intercalating drug complexes of very short chain oligonucleotides.

Studies have been made of base-pairing conformational parameters in single crystal structures of very short chain oligonucleotide structures complexed with drug molecules, using data extracted from the Cambridge structural database. The planar portion of the drug has a tendency to intercalate between two bases, utilising strong stacking interactions to stabilise the configuration. The effect of the existence of a formative backbone is seen in the high occurrence of standard base-pairing schemes and a consistent C1'-C1' separation, although the choice of compounds studied does tend to emphasise complementary pairing. In addition to the modulation of the general magnitude which is reduced from that in uncomplexed oligonucleotides, there appears to be some correlation of propeller twist value with the presence of planar groups sandwiching a base-pair. The average twist in such sandwiched pairs is lower than in any other group studied to date.