Advanced DNA assembly technologies in drug discovery

Abstract

Recombinant DNA technologies have had a fundamental impact on drug discovery. The continuous emergence of unique gene assembly techniques resulted in the generation of a variety of therapeutic reagents such as vaccines, cancer treatment molecules and regenerative medicine precursors. With the advent of synthetic biology there is a growing need for precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes. In this article, we summarize the highlights of the recombinant DNA technology since its inception in the early 1970s, emphasizing on the most recent advances, and underscoring their principles, advantages and shortcomings. Current and prior cloning trends are discussed in the context of sequence requirements and scars left behind. Our opinion is that despite the remarkable progress that has enabled the generation and manipulation of very large DNA sequences, a better understanding of the cell's natural circuits is needed in order to fully exploit the current state-of-the-art gene assembly technologies.

Keywords::

• cloning
• DNA assembly
• recombinant
• recombination
• synthetic biology

1. Introduction

This year marks the 40th anniversary of the pioneering work of Paul Berg's laboratory that resulted in the generation of the first recombinant DNA molecule [1]. Since then, molecular cloning has evolved using varying strategies and constituents and is now poised to be rebranded by the emerging synthetic DNA technology. Even though we will inevitably reach a stage where cloning will be replaced by fully automated DNA synthesis, the underlying principles of synthetic assembly will probably remain based on principles developed during the 1970s and 1980s.

Ever since human insulin, the first licensed drug generated using recombinant DNA technology was developed [2], drug discovery has been and continues to be profoundly reshaped by the developments on DNA assembly. Today, some of the drugs that used to be synthesized by organic chemistry or cumbersomely extracted from natural sources are now efficiently produced by complex synthetic pathways reconstituted in bacteria or other organisms (for a recent review see [3]).

In this article, we attempt to provide a high level assessment of the most recent and innovating ideas regarding DNA assembly, referring the reader to earlier reviews for further strategies, deeper information and extended references [4-8].

2. Sequence-dependent cloning

Before the emergence of the polymerase chain reaction (PCR) in the late 1980s, nearly all the cloning strategies were based on restriction/ligation protocols. These types of cloning procedures are sequence-dependent, as they rely on unique and specific sites in the target molecules. Other sequence-dependent assembly methodologies include site-specific recombination approaches such as Gateway® or Cre/LoxP, and topoisomerase cloning (for further information on these and other sequence-dependent strategies see [4]). Main providers for commercial products using the methodologies above are New England Biolabs (Ipswich, MA, USA), Agilent Technologies (Santa Clara, CA, USA) and Life Technologies (Carlsbad, CA, USA). Major shortcomings of these strategies are: i) the requirement of specific sites in the inserts and vector, and ii) the undesired operational sequences left behind, which could interfere with the function of the genetic assembly.

Nevertheless, restriction–ligation cloning has found an application that standardizes the DNA stitching process, the BioBrick^™ assembly [9]. Each BioBrick part is a DNA sequence flanked by two different and unique restriction sites, which allows the generation of larger BioBrick parts by chaining together smaller ones. The process creates junctions that lack the original sites. However, the use of a BioBrick part encoding a Type IIB restriction site partially overcomes the system's limitations in the sense that it allows for modification of already assembled circuits and scar removal [10].

The presence of undesired scars in the final construct has been addressed by several cloning approaches, including restriction-based strategies. One of these approaches, termed Golden Gate cloning [11], relies on the use of Type IIS restriction enzymes, whose recognition sites are distal from their cleavage sites, and therefore can be strategically placed in regions that may not be present in the final construct. The principle has proven useful for: i) shuffling repetitive sequences or gene variants [12] (prohibitive in procedures based on homologous recombination), ii) generating idempotent constructs as the BioBrick parts [13] and iii) producing multigene assemblies based on standardized modular cloning [14]. However, most of all these methodologies still rely on particular pre-existing target sites that must be absent in the individual parts.

3. Sequence-independent cloning

Due in part to the limitations imposed by the strategies described above, a number of methods that do not rely on specific target sites and do not leave unwanted sequences in the final construct have been developed. Most, if not all, are based on some form of the ‘chew back and repair' principle exploited by homologous recombination. The methods differ in the way the DNA fragments are produced and processed, but in all cases complementary single-stranded DNA ends become exposed and eventually annealed and repaired either in vitro or within the host organism. Examples of strategies based on in vitro processing include ligation-independent cloning (LIC) [15], sequence-and-ligation-independent cloning (SLIC) [16], uracil-specific excision (USER) [17], RNA-overhang cloning [18], PLICing [19], In-Fusion® assembly [20], pairwise selection assembly [21], circular polymerase extension cloning (CPEC) [22], isothermal/Gibson assembly [23] and GeneArt® Seamless and Cloning Assembly [24]. Several of these technologies have been turned into products commercialized by a variety of companies such as New England Biolabs, Lucigen (Middleton, WI, USA), EMD (San Diego, CA, USA), Clontech (Mountain View, CA, USA) and Life Technologies.

The principle above also applies to strategies where the processing of the DNA fragments occurs entirely within a living cell. For example, Escherichia coli cells that overexpress either the Rac prophage recET operon or the phage lambda redET genes (lambda red system) exhibit enhanced recombination proficiency [25]. The system efficiently achieves gene replacement and simple cloning reactions; however, it fails to assemble multiple fragments into a single construct (unpublished data). The system is commercially available from Gene Bridges (Heidelberg, Germany). The lambda red system may be used in combination with homing endonucleases and conjugal mating (MAGIC cloning), circumventing problems associated with ex vivo manipulation of DNA, but it depends on the use of specific vectors and engineered hosts [26]. Another organism used as a recombinogenic tool is Bacillus subtilis, where intermediate-sized DNA fragments are naturally transformed and ‘stitched' together in a stepwise manner generating large episomes or DNA structures integrated into the cell's chromosome [27].

But perhaps the most powerful ‘living' tool suitable for in vivo recombination is the budding yeast Saccharomyces cerevisiae. This organism has the unmatched ability to assemble and maintain constructs larger than 2 Mbp starting from dozens of overlapping DNA fragments of varying sizes with as few as 30 bp in common at their ends. The technology has been described as transformation-associated recombination (TAR) in the early 1990s and has been significantly improved over the last few years [28,29]. The system can also bridge two or more non-homologous DNA fragments using double-stranded ‘stitching' oligonucleotides [30]. This feature allows reusing existing fragments that had not necessarily been designed for a specific assembly, without having to re-amplify them to incorporate end homology. The approach can be taken to the extreme in the case where only oligonucleotides are employed as the building blocks, effectively utilizing yeast as a gene synthesis machine [31]. The system is commercialized by Life Technologies under the name of GeneArt® High-Order Genetic Assembly System. Finally, the recombinogenic properties of yeast have been harnessed by the ‘reiterative recombination' method [32]. The process is based on the expression of homing endonucleases that induce recombination at specific sites. Their use in combination with compatible yeast markers, allows an indefinite number of DNA fragments to be sequentially inserted at a defined locus.

4. Expert opinion

The DNA recombinant field has evolved to a point where two parallel trends are in place. On one side, there are the proponents of scar-based standardization of cloning assembly, who pursue the use of genetic prefixes and suffixes to pre-made fragments to guide the assembly in an idempotent manner and predetermined order. On the other side, the advocates of seamless cloning argue that in order to avoid undesired physiological effects genetic scars must be avoided. Standardization has yielded interesting, useful and relatively complex biological systems and holds the promise to open up genetic engineering to people from a variety of areas. However, it becomes cumbersome when applied to larger assemblies. Our view is that a combination of both approaches should result in a synergistic solution that ultimately will be left to gene synthesis providers or automated synthesizers.

During the last few decades, molecular cloning has had a fundamental impact on drug discovery. For example, various cloning techniques i) enabled the identification of specific receptor subtypes where traditional pharmacologic methods of using agonists and antagonists have failed, ii) permitted the development of the generation of antisense molecules, antibodies and small molecule peptides and iii) allowed the production of a variety of vaccines. More recently, with the advent of synthetic biology, the use of multigene assembly technologies empowered the generation of complex circuits required for the prevention of infections, cancer treatment and regenerative medicine among others [3].

By the redesign and reprogramming of existing biological systems, synthetic biology has underscored the need for robust systems to generate genome-sized assemblies. Proof-of-concept has been established [33]. However, there is still a technological gap between the ability to assemble large DNA molecules and to assemble large meaningful operating DNA. Today, we still struggle working with pathways on a mere 1 kb scale. Making substances that require the concerted expression of tens of genes may take years and significant funding. Many technologies must fall into place before we can fully exploit the remarkable advances of molecular cloning that have evolved over the past 40 years.

Declaration of interest

B Tsvetanova, L Peng, X Liang, K Li, TC Peterson and F Katzen are currently employed by Life Technologies. L Hammond works for Life Technologies as a field researcher.

Acknowledgements

The article may contain personal opinions and forward-looking statements, which may not reflect the opinions of Life Technologies. The GeneArt® High Order Genetic Assembly and GeneArt® Seamless Cloning and Assembly products are for Research Use Only (RUO), not for any human or animal therapeutic or diagnostic use.