Oligonucleotide are small DNA or RNA molecules, oligomers , that have wide applications in genetic testing , research and forensics . Usually made in the laboratory by solid-phase chemical synthesis , [1] these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and therefore can be used for artificial gene synthesis . , are important for polymerase chain reaction (PCR). ), DNA sequencing , molecular cloning and molecular investigationsin the form of, In nature, oligonucleotides are commonly found as small RNA molecules that function in the regulation of gene expression (such as microRNAs ), or as degradation intermediates derived from the breakdown of large nucleic acid molecules.
Oligonucleotides are characterized by a sequence of nucleotide residues that make up the entire molecule. The length of an oligonucleotide is usually denoted by a ” -mer ” ( from the Greek meros , “part”). For example, an oligonucleotide of six nucleotides (nt) is a hexamer, while one of 25 nt would usually be called a “25-mer”. Oligonucleotides readily bind to their respective complementary oligonucleotides, DNA, or RNA in a sequence-specific manner, forming duplexes or, less often, higher-order hybrids. This basic property is used to detect specific sequences of DNA or RNA .It serves as a basis for the use of oligonucleotides. Examples of procedures using oligonucleotides include DNA microarray , Southern blot , ASO analysis , fluorescent in situ hybridization (FISH), PCR , and synthesis of artificial genes .
Oligonucleotides are composed of 2′-deoxyribonucleotides (oligodeoxyribonucleotides), which can be modified at the backbone or at the 2′ sugar position to achieve various pharmacological effects. These modifications give new properties to oligonucleotides and make them a key element in antisense therapy .
synthesis
Oligonucleotides are synthesized chemically as building blocks, protected phosphoramidites of natural or chemically modified nucleosides, or to a lesser extent from non-nucleosidic compounds. The oligonucleotide chain assembly proceeds in the 3′ to 5′ direction by following a regular process referred to as a “synthetic cycle”. The completion of a single synthetic cycle results in the addition of a nucleotide residue to the growing chain. The yield of each synthetic step less than 100% and the occurrence of side reactions determine the practical limit of the efficiency of the process. In general, oligonucleotide sequences are usually short (13–25 nucleotides long). [4]The maximum length of synthetic oligonucleotides hardly exceeds 200 nucleotide residues. HPLC and other methods can be used to separate products with the desired sequence .
chemical modification
Creating a chemically stable short oligonucleotide was the earliest challenge in developing ASO treatments. Naturally occurring oligonucleotides are readily degraded by nuclease, an enzyme that cleaves the nucleotide and is sufficient in every cell type. [5] Short oligonucleotide sequences also have weak internal binding affinities, which contribute to their degradation in vivo.
spinal cord modification
Nucleoside organothiophosphate (PS) analogs of nucleotides give some beneficial properties to oligonucleotides. The main beneficial properties that give PS backbone nucleotides are the diastereomer identity of each nucleotide and the ability to easily follow reactions involving phosphorothioate nucleotides, which is useful in oligonucleotide synthesis. [7] PS backbone modification in oligonucleotides protects them from unwanted degradation by enzymes. [8] Modifying the nucleotide backbone is widely used because it can be achieved with relative ease and accuracy on most nucleotides. [7]Fluorescent modifications at the 5′ and 3′ ends of the oligonucleotides were reported to evaluate the oligonucleotides structures, dynamics and interactions with respect to the environment. [9]
sugar ring modification
Another modification that is useful for medical applications of oligonucleotides is the 2′ sugar modification. Modifying the 2′ position sugar increases the effectiveness of oligonucleotides by enhancing the targeted binding capabilities of the oligonucleotides, particularly in antisense oligonucleotides therapy. [6] They also reduce nonspecific protein binding, increasing the accuracy of targeting specific proteins. [6] Two of the most commonly used modifications are 2′-O-methyl and 2′-O-methoxyethyl. [6] Fluorescent modifications on the nucleobase were also reported.
Antisense Oligonucleotides
Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. [4] In the case of antisense RNAs, they inhibit the protein translation of certain messenger RNA strands by binding to them in a process called hybridisation. [10] Antisense oligonucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this hybrid can be degraded by the RNase H enzyme. [10] RNase H is an enzyme that hydrolyzes RNA, and when used in an antisense oligonucleotide application, causes 80–95% down-regulation of mRNA expression. [4]
The use of anti-sense oligonucleotides for gene knockdown in the morpholino backbone, which is now a standard technique in developmental biology and is used to study altered gene expression and gene function, first developed by Janet Heasman using Gone is Xenopus. [11] Morpholino drugs approved by the FDA include etaplirsen and golodirsen. Antisense oligonucleotides have also been used to inhibit influenza virus replication in cell lines.
Neurodegenerative diseases that result from a single mutant protein are good targets for antisense oligonucleotide therapies because of their ability to target and modify very specific sequences of RNA with high selectivity. Several genetic diseases, including Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), have been linked to DNA alterations that result in incorrect RNA sequences and result in mistranslated proteins that have toxic physiological effects. [14]
analytical techniques
chromatography
Alkalamides can be used as chromatographic stationary phases. [15] Those steps have been investigated for the dissociation of oligonucleotides. [16] Ion-pair reverse-phase high-performance liquid chromatography is used to separate and analyze oligonucleotides after automated synthesis. [17]
mass spectrometry
A mixture of 5-methoxysalicylic acid and spermine can be used as the matrix for oligonucleotides analysis in MALDI mass spectrometry. [18] Electrospray ionization mass spectrometry (ESI-MS) is also a powerful tool to characterize the mass of oligonucleotides. [19]
DNA microarray
DNA microarray is a useful analytical application of oligonucleotides. Compared to standard cDNA microarrays, oligonucleotide-based microarrays have more controlled specificity upon hybridization, and have the ability to measure the presence and spread of alternatively spliced or polyadenylated sequences. [20] A subtype of DNA microarrays can be described as substrates (nylon, glass, etc.), in which oligonucleotides are bound at high densities. [21] DNA microarrays have many applications in the life sciences.
