Transfection: the delivery of foreign nucleic acids into a cell. The possible applications for transfection seem endless. But… how exactly does TransIT® transfection work? In today’s post, we’ll discuss what transfection complexes are and how nucleic acid cargo gets into cells.
Chemical transfection of nucleic acids provides a convenient and robust alternative to viral, liposomal encapsulation and electroporative delivery. At Mirus we design, synthesize, formulate and test our TransIT® reagents to deliver any nucleic acid to a broad range of cell types, including hard-to-transfect cells. While optimization of the formulation and structure of a transfection reagent is central to how Mirus develops its range of novel TransIT® reagents, other aspects such as physical properties of resulting nucleic acid complexes are equally important. These complexes are influenced by several factors, which is why Mirus includes easy-to-follow transfection complex formation protocols with each product.
A transfection complex forms when nucleic acids are combined with one of our TransIT® reagents. Through a combination of electrostatic and other noncovalent interactions, our cationic non-viral, non-liposomal formulations bind the negatively charged nucleic acids to stabilize and condense them, enabling cell surface binding. The resulting transfection complexes are usually several hundred nanometers in diameter. When incubated with cells, the positively charged complexes electrostatically interact with the negatively charged cellular membrane, enabling cellular uptake through endocytosis. Once endocytosed, transfection complexes can become trapped in endosomes, potentially leading to the undesired outcomes of lysosomal degradation or cellular export. Desired changes in gene expression will not occur unless the nucleic acid escapes the endosome and interacts with cellular machinery in the cytoplasm (e.g. for siRNA and mRNA transfections) or nucleus (e.g. for DNA or CRISPR guide RNA transfections).
Therefore, Mirus TransIT® reagents have been designed to effectively overcome each of these cellular barriers; however, the physical properties of transfection complexes that evolve during their formation can drastically affect their success.
For a quick overview of the transfection process, watch an animated illustration “The Simplicity of Transfection.”
Physical properties, such as size and charge of transfection complexes, can vary drastically and depend on several factors:
These factors should all be considered when forming your transfection complexes and underpin the reasoning for the protocols described for each individual transfection reagent. At Mirus Bio, we are known as the Transfection Experts because we perform many chemical, materials science and biological experiments to optimize nucleic acid delivery for a diverse range of applications.
We study transfection complexes under a wide variety of conditions to correlate their chemical and physical properties with their biological behavior. These complex properties, primarily size and charge, are in turn heavily influenced by the conditions under which the complexes are formed. Our chemists employ advanced characterization techniques such as dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta-potential (ζ-potential) measurement to measure transfection complex size and surface charge. Certain characteristics of complexes often need to be present for the complex to be efficacious: namely the complex should be within a certain size range and should have a net positive charge. Understanding aspects of how the complex behaves allows us a better chance of correlating these behaviors with optimal functional delivery for different cell types and applications.
Through testing of various TransIT® transfection reagents in multiple cell lines, we have observed that some transfection reagent formulations perform well in certain cell lines and poorly in others (see Reagent Agent®). While successful transfection is typically only assessed through reporter gene expression, on a more fundamental level, selecting the right transfection reagent is about finding a match between the physical/chemical properties of the transfection complex and the endomembrane system of the target cell.
To demonstrate how following our recommended protocols maximizes transfection efficiency, we have highlighted how performing key protocol steps are as important as choosing the correct transfection reagent for your cell line.
The composition of the transfection reagent affects the size of the resulting complex post addition of nucleic acid. The differences in size (radii ranging from 300 nm to 750 nm) and resulting transfection efficiency of complexes formed using different formulations of transfection reagent is illustrated below. In this experiment performed in HEK 293-F cells, transfection reagents that formed complexes below ~400 nm resulted in higher transient transfection expression levels compared to the transfection reagents that formed larger complexes. It is important to note that a size of below 400 nm does not always result in high transfection efficiency; it is a necessary but not sufficient experimental parameter.
Complex Size versus Transfection Efficiency for Various Transfection Complex Formulations. After incubating each transfection reagent with plasmids for 15 min, the size of the transfection complex was determined with DLS. HEK 293-F cells were transfected with each complex and relative transfection efficiency was determined.
Dynamic growth of transfection complexes is observed over time (see Figure below). Typically, a transfection complex with a radius of 200-400 nm is optimal for most applications. Complex formation time is one of the main parameters that researchers can vary to control transfection complex size in order to achieve successful nucleic acid delivery. For our TransIT® transfection reagents, we have determined the optimal complex formation time (and corresponding complex size) to achieve the intended results in the cell type or application for which the reagent was developed. These optimal complex formation times are specified in each TransIT® transfection reagent protocol. For example, the recommended TransIT®-mRNA transfection complex formation time is < 5 min, while that of TransIT-X2® is 15-30 min.
Transfection Complex Size Increases over Time in Complex Formation Medium. Transfection complexes were formed by mixing a transfection reagent with plasmid at a 3:1 reagent-to-DNA ratio. The size of the transfection complex was measured with DLS at the indicated times after mixing.
Like complex formation time, the absolute reagent and nucleic acid concentration can also influence the dynamics of transfection complex growth (see Figure belwo). In this example, the growth of complexes was accelerated in more concentrated reagent and DNA mixtures. As such, our recommended reagent and nucleic acid concentrations for each reagent complement their respective recommended complex formation times to yield the highest transient transfection expression levels. For example, we recommend using 1 µg DNA and 1 µl of TransIT-PRO® Transfection Reagent per 0.1 ml of complex formation solution for suspension CHO cell transfections. The recommendations provided in our protocols are good starting points. In some cases, optimization around these recommended concentrations can be performed to further improve on transfection efficiency depending on your specific cell type or application. Accordingly, for protein production in suspension HEK 293 cells, optimal yields were reported at varying transfection complex concentrations ranging from 1-2 µg DNA and 1-2 µl of TransIT-PRO® Transfection Reagent per 0.1 ml of complex formation solution.
Growth of Transfection Complexes Is Affected by Concentration of Transfection Reagent and DNA. Transfection complexes were formed by mixing a transfection reagent with plasmid at the same reagent-to-DNA ratio (3:1) but at three different concentrations, i.e. 3:1, 2.25:0.75, 1.5:0.5 (µl:µg) for 1X, 0.75X, and 0.5X, respectively. The size of the transfection complex was measured with DLS at the indicated times after mixing.
At Mirus, our protocols recommend a certain volume of our reagent be added to a corresponding mass of nucleic acid, i.e. recommended reagent-to-DNA ratio. The molar charge ratio of cationic TransIT® reagent to anionic nucleic acid determines the overall charge of the transfection complex. The molar charge ratio also dictates how these oppositely charged species will interact and aggregate. The Figure below demonstrates how the reagent-to-DNA ratio can affect transfection complex growth. In addition to complex formation time, the ratio of reagent to nucleic acid is another parameter that researchers can vary to influence transfection complex size. And, as discussed above, transfection complex size is a key component for transfection success. Note, each TransIT® reagent can exhibit a different complex growth profile for a given reagent-to-DNA ratio. Furthermore, the optimal ratio may differ between cell types and applications, thus our recommendation to empirically determine the optimal reagent-to-DNA ratio (PDF) for each new cell type and or application OR to utilize pre-existing information, when available, in our Reagent Agent® transfection database.
Growth of Transfection Complexes for Two Different Reagent-to-DNA Ratios. Transfection complexes were formed by mixing a transfection reagent with plasmid at a 3:1 (red) or 1:1 (black) reagent-to-DNA ratio. The size of the transfection complex was measured with DLS at the indicated times after mixing.
Mirus recommends forming transfection complexes in buffered, saline solutions such as Opti-MEM® or, in some cases, PBS or other serum-free basal media. Serum contains negatively charged proteins, which can inhibit complex formation, and nucleases, which can degrade the nucleic acid cargo prior to being protected by the transfection reagent. Generally, transfection complexes consisting of multiple copies of the nucleic acid cargo must aggregate to a certain size to achieve effective transfection. When utilizing cationic lipid and polymer transfection reagents in the absence of salt, this aggregation of complexes does not occur (see Water and HEPES conditions, Figure below). The pH of the complex solution can also affect the dynamics of transfection complex formation (compare complex growth in Opti-MEM® at pH 7.4 versus pH 8.5, Figure below). Taken together, the solution in which you form your transfection complexes is critical to the success of your transfection.
Growth of Transfection Complexes in Various Complex Formation Media. Transfection complexes were formed in the indicated media. The size of the transfection complex was measured over time with DLS.
Delving deeper into the structure of our TransIT® reagents, the Figure below shows how the polymer structure of a transfection reagent can affect transfection complex size. In some cases, as in this example, the size of the transfection complex increases with polymer chain length. Note, complex size does not necessarily correlate with polymer length for all transfection reagents. Polymer length is just one of several variables studied at Mirus Bio during development of novel, effective transfection reagents.
Effect of Reagent Polymer Length on Transfection Complex Size. Transfection complexes were formed by incubating DNA with transfection reagents of varying polymer lengths. After 15 min, the size of each transfection complex was measured with DLS.