Oligonucleotides are short, synthetic strands of nucleic acids. The term “oligo” derives from a Greek word meaning “few” or “small,” reflecting their limited length compared to natural nucleic acids. These molecules are manufactured in laboratories, rather than being isolated from biological sources. Understanding their structure is key to understanding their diverse applications in scientific research, diagnostics, and therapeutic approaches.
Basic Building Blocks
The fundamental units that construct an oligonucleotide are called nucleotides. Each nucleotide has three parts: a five-carbon sugar, a phosphate group, and a nitrogen-containing base.
The sugar component is either deoxyribose for DNA-based oligonucleotides or ribose for RNA-based ones. The phosphate group attaches to the 5′ carbon of the sugar. The nitrogenous base, which is the information-carrying part of the nucleotide, is attached to the 1′ carbon of the sugar.
There are four types of nitrogenous bases in DNA, with a variation in RNA. These bases are adenine (A), guanine (G), cytosine (C), and thymine (T). In RNA, uracil (U) replaces thymine (T). These bases are categorized into two groups based on their chemical structure: purines, which have a double-ring structure, include adenine and guanine, while pyrimidines, with a single-ring structure, include cytosine, thymine, and uracil.
Assembling the Oligonucleotide Chain
Individual nucleotides connect to form a single oligonucleotide strand through chemical bonds. This linkage creates a continuous backbone. The sugar and phosphate components of adjacent nucleotides form the repetitive sugar-phosphate backbone of the strand.
The connection between nucleotides occurs via phosphodiester bonds. The phosphate group attached to the 5′ carbon of one nucleotide’s sugar forms a bond with the hydroxyl group on the 3′ carbon of the next nucleotide’s sugar.
This linking establishes the oligonucleotide chain’s directionality, known as the 5′ to 3′ (five-prime to three-prime) direction. One end of the chain will have a free phosphate group at the 5′ carbon, known as the 5′ end, while the other end will have a free hydroxyl group at the 3′ carbon, known as the 3′ end. This directionality influences how oligonucleotides interact with other nucleic acids and proteins, affecting their biological function and synthetic assembly.
Single and Double Stranded Structures
Oligonucleotides can exist as single strands or interact with other nucleic acid strands to form double-stranded structures. When two complementary single strands come together, they form a double-stranded structure through hybridization. This interaction is specific, relying on complementary base pairing.
In complementary base pairing, adenine (A) pairs with thymine (T) in DNA, or with uracil (U) in RNA. Guanine (G) pairs with cytosine (C). Hydrogen bonds mediate these pairings: two hydrogen bonds form between A and T/U, while three hydrogen bonds form between G and C. These hydrogen bonds, though individually weak, collectively provide stability to the double-stranded structure.
Several factors influence the stability of these double-stranded oligonucleotide structures. The length of the oligonucleotide influences stability, with longer sequences forming more stable duplexes due to more hydrogen bonds. The GC content, the proportion of guanine-cytosine pairs, also impacts stability because G-C pairs form three hydrogen bonds compared to two in A-T pairs, making GC-rich regions more stable. Temperature is another factor, as higher temperatures disrupt hydrogen bonds, leading to strand separation (denaturation).
Oligonucleotides Compared to DNA and RNA
Oligonucleotides share a similar chemical foundation with DNA and RNA but differ significantly in their overall size and typically their origin. The most notable distinction lies in their length; oligonucleotides are considerably shorter, generally consisting of tens to a few hundred nucleotides. In contrast, naturally occurring DNA and RNA molecules are polynucleotides, meaning they are much longer, often composed of thousands to millions of nucleotides.
While both are nucleic acids, the specific sugar component and one of the nitrogenous bases can vary depending on whether the oligonucleotide is designed to mimic DNA or RNA. DNA-based oligonucleotides contain deoxyribose sugar and utilize thymine as one of their bases. RNA-based oligonucleotides, however, feature ribose sugar and use uracil in place of thymine. These subtle chemical differences influence the stability and flexibility of the resulting strand.
Furthermore, oligonucleotides are predominantly synthetic molecules, precisely manufactured in laboratories with user-defined sequences for specific applications. While some short RNA molecules exist naturally in biological systems, such as microRNAs, the vast majority of oligonucleotides used in research and therapeutics are custom-made. This contrasts with large DNA and RNA molecules, which are typically isolated from living organisms.