The Tn5 transposase enzyme, a modified version of Transposon 5 (Tn5), has become a foundational tool in modern molecular biology. This enzyme is a naturally occurring “jumping gene” that moves and inserts copies of itself into different locations within a genome. The ability of this engineered enzyme to precisely cut DNA and simultaneously attach short sequence tags has streamlined the process of preparing DNA for sequencing, accelerating genomic research and discovery. The enzyme’s unique mechanism enables new methods that provide insights into how the genome is structured and regulated.
The Bacterial Origin of Transposon 5
Transposon 5 is a composite mobile genetic element, a type of DNA sequence that can change its position within the genome of a cell. It was identified in the bacteria Escherichia coli. The Tn5 element is composed of a central region flanked by two near-identical insertion sequences, known as IS50L and IS50R, in an inverted orientation.
The central region typically carries genes that confer antibiotic resistance, such as the ability to resist kanamycin and streptomycin. This genetic cargo is one reason Tn5 has been successful, as its movement can quickly transfer drug resistance traits between different parts of a bacterium’s genome or to other bacteria. The flanking IS50 elements contain the genetic code for the Tn5 transposase enzyme, the molecular machine responsible for the element’s movement.
The transposase protein recognizes specific 19 base-pair sequences at the ends of the IS50 elements, referred to as the outside ends. This recognition allows the enzyme to bind to the ends and prepare the element for its move. The natural role of Tn5 is to ensure its own propagation and the dispersal of the beneficial genes it carries.
The Cut and Paste Mechanism of Tn5
The movement of the Tn5 element occurs through conservative transposition, commonly known as the “cut and paste” mechanism. The element excises itself completely from its original location before inserting into a new site. The process begins when two molecules of the transposase enzyme bind to the inverted repeats at the ends of the Tn5 element, forming a dimeric protein-DNA complex called a synaptic complex.
Within this complex, the enzyme coordinates divalent metal ions, typically magnesium, which are necessary for the catalytic reaction. The transposase then performs a staggered cut at the ends of the element, excising the Tn5 DNA entirely from the donor site.
The excised element, still bound to the transposase dimer, is then guided to a new, non-specific location in the host DNA. The enzyme performs a nucleophilic attack on the target DNA backbone, inserting the transposon and creating a gap on either side. This insertion results in a characteristic 9 base-pair duplication of the target DNA sequence, which is later filled in by the host cell’s repair machinery.
Repurposing Tn5 for Laboratory Use
To transform the Tn5 enzyme into a powerful laboratory tool, scientists introduced specific amino acid substitutions to create a “hyperactive” mutant. These genetic modifications, such as the E54K and L372P mutations, significantly increase the enzyme’s efficiency and speed of transposition.
The major innovation that repurposed Tn5 for genomics was the development of the “tagmentation” technique. This process leverages the enzyme’s natural ability to cut DNA and insert a sequence, but with a modification. The transposase enzyme is pre-loaded with synthetic, double-stranded DNA oligonucleotides, known as adapters. These adapters contain sequences needed for subsequent sequencing steps.
When the hyperactive Tn5 enzyme, bound to these adapters, encounters a target DNA molecule, it performs its cut-and-paste action. Instead of inserting a full transposon, the enzyme simultaneously fragments the target DNA and attaches the pre-loaded sequencing adapters to the ends of the newly created fragments. This single-step reaction replaces several labor-intensive steps previously required for preparing DNA sequencing libraries.
Accelerating Genomics with Tn5 Tagmentation
Tn5 tagmentation has accelerated the process of Next-Generation Sequencing (NGS) library preparation, making it faster and more efficient. The single-tube reaction simplifies the workflow, allowing researchers to process a larger number of samples with less hands-on time and reagent cost. The technique is valuable because it requires very small amounts of starting material, sometimes even sub-picogram quantities of DNA, enabling studies on precious or limited biological samples.
The most important application of this technology is the Assay for Transposase-Accessible Chromatin using sequencing, or ATAC-seq. This method uses the Tn5 transposase to map regions of “open chromatin” in the genome. These are areas of the DNA that are not tightly packed around proteins and are accessible to the cell’s gene-reading machinery.
The Tn5 enzyme preferentially inserts its adapters into these open, regulatory regions, allowing scientists to quickly and precisely identify the parts of the genome that are actively involved in gene expression. Other applications include mapping protein-DNA interactions (CUT&Tag) and analyzing genomic variations.