This month we are celebrating a small thing—NanoLuc® luciferase, an enzyme whose tiny size and bright luminescence enable much more sensitive detection of intracellular events than other bioluminescent reporters. Scaling down the size of the luciferase protein makes it “fit” in situations where larger reporters do not, and also makes it less likely to interfere with natural biology than larger proteins. Scaling up the brightness allows you to detect the reporter at low abundance, enhancing sensitivity and allowing detection of small changes in gene expression at concentrations closer to physiological levels.
These properties of Nanoluc® luciferase allow its bioluminescence to be used to detect intracellular events in ways not possible before. One example of how this small size and intense brightness are being applied to help solve biological problems is the insertion of NanoLuc® luciferase into influenza viruses, where the genome size is small and does not tolerate large insertions. Unlike viruses incorporating larger luciferases, the influenza reporters incorporating NanoLuc® luciferase are stable, retain pathogenicity and are bright enough to track at low doses during the early stages of infection (1). You can find out more about the many applications of NanoLuc® luciferase here.
In the spirit of “celebrating small things” we will be sharing plenty of information about NanoLuc® applications over the next few weeks, and will also be highlighting other examples where “small” can be a beautiful thing. By definition, molecular biology is a study of the smallest building blocks of life, so it’s not hard to find an abundance of small things to talk about. We hope you will participate with us by commenting, liking or sharing posts, or by contributing your own ideas on little things that make a difference, or that just make you smile.
Here’s a list for starters—it includes examples from history and from last week where going smaller turned out to be both smarter and better for overcoming a variety of biological challenges.
- Smaller, Simpler Organisms: Solving the Challenge of Complexity
Bacteria, phage and plasmids—On these little things the whole field of molecular biology rests. Classic studies on bacteria, phage and plasmids were used to answer fundamental questions about the nature of DNA. Where would we be without E. coli, lambda and T4? Through studies on these simple organisms, the science of molecular biology came into existence. Manipulation of DNA in bacteria, phage and plasmids enabled many “firsts” including identification of DNA as the “heritable material”, identification of restriction/modification systems, and cloning of genes. Later, the first genomes sequenced were those of phage, then bacteria. Use of these simple organisms with small genomes allowed the development of model systems on which many of the things we take for granted today were first worked out experimentally.
- Nanotechnology: Solving the Challenge of Size
Advances in molecular biology coupled with the ability to manipulate materials at molecular scale have led to the development of promising new avenues of treatment for cancers (2). Nanotechnology based treatments offer hope for precision destruction of targeted tumor cells while leaving normal cells intact. Nanoparticles incorporating surface peptides or antibodies targeting specific tumor cells have been developed, and several are the subject of clinical trials. They can be used to deliver drugs, or can be activated by radiation. Nanoshells are an example of the latter. These tiny silica particles are covered in gold, onto which antibodies targeting specific cell types can be attached. Once in situ, the gold surface is superheated by radiation, killing the surrounding cells (3).
- Nanotechnology: Solving the Challenges of Throughput and Scale
The newest next-gen sequencing methods incorporate nanoscale chambers and nanopores to interrogate single DNA molecules, offering much higher throughput, faster sequencing, longer read lengths and reduced cost compared to PCR-based methods. In these systems, nanometer-scale pores or chambers are used to isolate single DNA strands (4). As the strand passes through the nanopore, the different bases cause characteristic disruptions in current, allowing real-time sequencing. Availability of nanoscale technologies such as these continues to revolutionize next-gen sequencing capabilities—making whole organism sequencing a viable option for many situations including epidemiological investigations, tissue typing and food testing, to name but a few.
- A Mini-Enzyme for Gene Editing: Solving the Challenge of Deliverability
CRISPR technology for precise gene editing has been in the news for a couple of years now. This gene editing technique provides a way to cut DNA very specifically using the bacterial protein Cas9 (derived from Streptococcus pyogenes), linked up to a target-specific RNA. The technology has been used by many groups to perform gene editing in a variety of species, giving hope that it may eventually enable specific genome editing to remove deleterious sequences from human DNA. One barrier to application of genome editing in vivo has been that the size of the Cas9 enzyme is too large to be packaged into the viral delivery system used to deliver modified genes to human cells. A paper published last week in Nature describes how one group of researchers used a smaller Staph aureus-derived Cas9 enzyme and were able to successfully package it into a viral delivery system and deliver modified genes to mouse cells (5).
Follow our small celebration on Facebook, Twitter or LinkedIn during April. We’ll be using the hashtag #nanoluc to share more information on the applications of NanoLuc luciferase, and also to have some fun celebrating the little things that make a difference in science and in our lives.
- Tran, V., et al. (2013) Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread. J. Virol. 87, 13321–9.
- Sun, T., et al. (2014) Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angew. Chem. Int. Ed. 53, 12320–64.
- NanoPore Detectors
- Brennan, Z. (2014) Gold ‘nanoshells’ may offer new delivery option for cancer, hypothermia drugs.
- Ran, F.A. et al. (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature published online 1 April. doi:10.1038/nature14299