Oligonucleotides

Oligonucleotides are short polymers composed of nucleotides linked by phosphodiester bonds. They are typically DNA or RNA, ranging from about 2 to 200 nucleotides in length, with common laboratory applications using 15–25 bases for primers or probes. Oligonucleotides can be single-stranded or double-stranded and may carry chemical modifications to improve stability, affinity, or cellular uptake.

Most oligonucleotides are synthesized chemically by stepwise phosphoramidite coupling on a solid support. The sequence determines base pairing and information content. The backbone is normally phosphodiester; modifications such as phosphorothioate linkages, 2'-O-methyl, 2'-fluoro, or locked nucleic acids (LNA) can enhance nuclease resistance and binding affinity. End-blocking groups or fluorophores can be added for detection or to block extension.

After synthesis, oligonucleotides are deprotected and purified, commonly by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis. Quality control includes determining identity, purity, and concentration by UV spectroscopy and often mass spectrometry.

Applications span research, diagnostics, and therapeutics. In research, DNA oligos serve as primers for polymerase chain reaction (PCR), sequencing, and DNA probes for hybridization assays; RNA oligos are used in RNA research and as crRNAs in certain CRISPR systems. In diagnostics, oligos act as probes in microarrays and in situ hybridization. In medicine, antisense oligonucleotides (ASOs), small interfering RNA (siRNA), and other modified oligos are investigated or approved for gene-silencing therapies; challenges include delivery, off-target effects, and stability.

Oligonucleotides are versatile, modular agents for information storage, recognition, and regulation of genetic material, with broad uses in basic biology, diagnostics, and therapeutics.