Oligonucleotides: Design and Applications



Oligonucleotides (or oligos) have many uses from research to disease diagnosis and recently therapeutics. The wide availability of inexpensive synthetic single-stranded DNA and RNA has opened the way for routine DNA amplification by PCR. This use of oligonucleotides as primers has revolutionized the study of gene expression and disease processes. There is an increasing array of primer-based technologies for characterizing, tracking, and measuring anything that involves nucleic acids directly or indirectly. In addition to their use as PCR primers, oligonucleotides are used as probes, in microarray, in situ hybridization, and antisense analyses, and even as drug carriers.

Related Topics: qPCR/Real-Time PCR Reagents, qPCR Assay Design and Optimization and PCR Primer and Probe Chemistries.

Types of Oligonucleotides

Oligonucleotides are short, single-stranded polymers of nucleic acid. Oligos may be unmodified or modified with a variety of chemistries depending on their intended use, for example, the addition of 5' or 3' phosphate groups to enable ligation or block extension, respectively, labeling with radionuclides or fluorophores and/or quenchers for use as probes, the incorporation of thiol, amino, or other reactive moieties to enable the covalent coupling of functional molecules such as enzymes, and extension with other linkers and spacers of diverse functionality. DNA oligos are the most commonly used, but RNA oligos are also available. The length of an oligo is usually designated by adding the suffix -mer. For example, an oligonucleotide with 19 nucleotides (bases) is called a 19-mer. For most uses, oligonucleotides are designed to base-pair with a strand of DNA or RNA.

PCR Primers

The most common use for oligonucleotides is as primers for PCR (polymerase chain reaction). Primers are designed with at least part of their sequence complementary to the 5' end of the sequence targeted for amplification.

The following characteristics are desirable for efficient PCR primers:

  • Optimal length for a complementary sequence is 18–22 nucleotides
  • Paired primers must have similar melting temperatures (Tm)
  • Optimal composition is 40–60% GC with a GC clamp (a pair of G or C bases) within the last 5 bases of the 3' end and no more than 3 Gs or Cs should be in the last 5 bases at the 5' end
  • Long repeats of a single base should be avoided
  • Few or no secondary structures such as hairpins
  • No self-dimers; a primer should not be homologous to itself
  • No cross dimers; primer pairs should not dimerize

Optimal primer sequences for PCR are usually determined by primer design software. For some targets, it is not easy to find a pair of primers that meet the above criteria even with software design help.

There are a number of primer designs with different primer- and probe-based detection chemistries for fluorescent detection of target amplification. Most fluorescent probes use fluorescence quenching, in which a fluorescent reporter is quenched by the close proximity of a quencher until the primer hybridizes to a single strand of nucleic acid. Depending on the probe design, when a primer binds to its complementary sequence, either the distance between the reporter and quencher is increased or the reporter is cleaved from the primer. The reporter is then able to fluoresce. Fluorescent probes can be used for accurate quantitation of gene expression by real-time PCR or digital PCR.

PCR Assays and Panels for Biologically Related Genes

In many areas of research, PCR assays and panels have been designed to detect and measure the expression of genes that are biologically related. Preconfigured assay plates are available for an increasing range of canonical pathways, diseases, signaling and other cellular processes, with examples including the IL-12 signaling pathway, glycogen metabolism genes, and groups of proteins such as growth factors.

Bio-Rad's laboratory-validated PrimePCR™ assays (currently available in the US and Canada only) include preconfigured assay panels covering complex interacting pathways that can reveal patterns of biologically related gene regulation and interaction, providing a comprehensive picture of signaling, metabolic, and disease processes. These panels are designed to measure changes in the expression of many genes simultaneously. These oligonucleotides are designed so that the PCR amplicons

  • Avoid regions containing SNPs
  • Span introns where possible
  • Detect the maximum number of transcription variants
  • Avoid areas of cross-homology with other targets


Modern sequencing employs the same basic technology as PCR, with the binding of a primer to single-stranded DNA followed by extension, although the different platforms use different technologies to read the resulting sequence of bases. Wherever possible, universal primers are used rather than target-specific primers. Universal primers with sequences complementary to those flanking the multiple cloning site (MCS) of the carrier plasmid can be used to sequence DNA that has been cloned into common plasmids. In next-generation sequencing, oligonucleotides are ligated onto the ends of all the nucleic acid fragments to be sequenced, and a universal primer is then used for sequencing.

DNA Microarrays

Microarrays have many microscopic spots of DNA, usually oligonucleotides, bound on a solid support. Assay targets can be DNA, cDNA, or cRNA. Depending on the system, the hybridization of targets to specific spots is detected by fluorescence, chemiluminescence, or colloidal silver or gold. Microarrays are used for multiple applications such as simultaneous measurement of the expression of large numbers of genes, enabling genome-wide gene expression analysis, as well as genotyping studies using single-nucleotide polymorphism (SNP) analysis.

Fluorescence In Situ Hybridization (FISH)

FISH is an important tool for detecting and localizing either DNA or RNA within cells and tissues. Either fragments of DNA or oligonucleotides can be used as probes. Oligonucleotides for FISH typically have the following characteristics:

  • Generally 20–30 nucleotides long
  • Can be directly labeled with fluorescent dyes such as Cy3 or Cy5
  • Labeling with digoxigenin or biotin is usually paired with fluorescent secondary antibodies
  • Labeled at ends or internally at multiple sites to increase sensitivity
  • Multiple, nonoverlapping oligonucleotides are often used

After hybridization of the probes to the target, fluorescence microscopy is used to determine the localization of probes and in some cases estimate the level of fluorescence. FISH is used in both basic research and clinical studies; it is used extensively in pathology.

Antisense Oligonucleotides

Antisense oligonucleotides are used to reduce levels of protein synthesis by inhibiting mRNA processing or translation. This approach is the basis of many therapies that are now in clinical trials including a range of cancer treatments. Antisense therapy is currently available for cytomegalovirus retinitis and familial hypercholesterolemia.

An antisense oligonucleotide has a sequence that is complementary to a sequence within a specific mRNA, resulting in the formation of a short double-stranded section. Antisense oligonucleotides can be unmodified or modified in one of several different ways. Whether an oligonucleotide is modified determines the mechanism(s) of inhibiting protein synthesis, the ability of the oligonucleotides to enter cells, and their susceptibility to degradation.

Inhibition mechanisms are:

  • RNase H degradation of the double-stranded section of mRNA (unmodified)
  • Double-stranded section stalls the translation machinery (all)
  • Blocking of RNA splicing proteins or binding of ribosomal initiation complex (morpholinos)

Currently, for most therapeutic antisense applications, morpholino oligonucleotides targeted to sequence upstream of the AUG translation start site, or within 30 base pairs downstream of the AUG, are the most effective.

Other Uses of Oligonucleotides

There are many other uses of oligonucleotides in research and therapeutics. Three of the most common applications are aptamer design, allele-specific testing, and triplex-forming oligonucleotides for dsDNA binding.

Nucleic Acid Aptamers: Aptamers are sequences of either DNA or RNA that bind with high specificity and affinity to a target, usually a protein. The specificity is due to the tertiary structure (helices, etc.) not the sequence per se.

Aptamers are selected by rounds of enrichment of random oligonucleotides or library fragments in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are often modified to increase stability; nanoparticles and drugs can be conjugated to aptamers.

Aptamers are used as sensors, regulators of cellular processes, and therapeutic tools. The high-affinity binding of aptamers can block protein function. Aptamers that bind to cell surface proteins can be used for drug delivery. There are many aptamers, mainly RNA aptamers, in clinical trials for diseases including cancer, macular degeneration, and clotting disorders.

Allele-Specific Oligonucleotides (ASO): ASO probes are used primarily in dot-blots for the detection of genetic polymorphisms. The oligonucleotides are generally 15–20 nucleotides long and are mainly used in testing for diseases with common point mutations, for example, cystic fibrosis and sickle-cell anemia. The binding of two oligonucleotides, one with the wild-type sequence and the other with the mutation, is compared to determine whether or not the mutation is present in a sample.

Triplex-Forming Oligonucleotides (TFOs): TFOs are oligonucleotides that fit in the major groove of DNA. They are generally 10–30 nucleotides long and bind to DNA sequences that have a long sequence of purines on one strand and of pyrimidines on the other. TFOs can reduce the expression of a gene by blocking transcription and are also being investigated for gene modification in targeted recombination to produce heritable changes to DNA.

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