In the Literature

Blogs about published peer-reviewed scientific studies.

Reviewing the Importance of circRNA

Posted on by Kerstin Hurd

In recent years following the COVID-19 pandemic, RNA has gained attention for its successes and potential use in vaccines and therapeutics. One avenue of interest in RNA research is a non-coding class of RNA first identified almost 50 years ago, circular RNA (circRNA).

In 1976, Sanger et al. first identified circRNA in plant viroids, and later additions to the field found them in mice, humans, nematodes, and other groups. Unlike linear RNA, circRNA are covalently closed loops that don’t have a 5′ cap or 3′ polyadenylated tail. Following its discovery, researchers thought circRNA was the product of a rare splicing event caused by an error in mRNA formation leading to low interest in researching the subject (1).

In the early 2010s, following the development of high throughput RNA sequencing technology, Salzman et al. determined that circRNAs were not a result of misplicing, but a stable, conserved, and widely sourced form of RNA with biological importance. Since noncoding RNA makes up the majority of the transcriptome it’s an incredibly important field of study. We now recognize circRNAs for their potential as disease biomarkers and importance in researching human disease (2).

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For Frogs, Surviving the Heat Could Come Down to What Is in Their Gut

Posted on by Kelly Grooms

Amphibians are the most threatened vertebrate class worldwide. Because they lack the ability to regulate their own temperature and moisture levels, climate change is playing a significant role in this growing peril (1). Climate change impacts amphibian survival in several ways. In addition to habitat loss, growing drought conditions make maintaining body moisture levels challenging and warming temperatures restrict activity periods needed for reproduction as well as increasing the risk of heat stress.

Heat tolerance varies by species, and understanding what influences these differences could help predict species survival. The gut microbiota is known to affect a wide range of functions in host animals, and recently studies have begun to investigate its role in host thermal tolerance (2).

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Treating Solid Tumors: Combining CAR-T Cell Therapy with Probiotics

Posted on by Johanna Lee

Chimeric Antigen Recepter (CAR)-T cell therapy is a personalized immunotherapy that harnesses the patient’s own immune system to combat cancer. It is done by engineering the patient’s T cells to specifically target and attack cancer cells in their body, and it has shown great success in treating various blood cancers such as leukemia.

Treating solid tumors with CAR-T cells, however, has proved much more challenging. This is mainly because solid tumors contain a heterogeneous population of cells, expressing a variety of antigens—many of which are also expressed in healthy cells. Therefore, T cells targeting solid tumors could potentially attack healthy tissue, resulting in serious side effects. In addition, solid tumors create a hostile microenvironment that is difficult for CAR-T cells to infiltrate.

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Glowing Testimonies: A Review of NanoLuc® Use in Model Organisms

Posted on by Kerstin Hurd
NanoLuc®

Model organisms are essential tools in the pursuit of understanding biological processes, elucidating the mechanisms of diseases, and developing potential treatments and therapies. Use of these organisms in scientific research has paved the way for groundbreaking discoveries across various fields of biology. In particular, non-mammalian models can be valuable due to characteristics such as rapid life cycles, low cost, and amenability to use with advanced genetic tools, including bioluminescent reporters such as NanoLuc® Luciferase.

NanoLuc® is a small (19.1 kDa) luciferase enzyme originating from deep sea shrimp that is 100x brighter than firefly or Renilla luciferase. It utilizes a furimazine substrate to produce its bright glow-type luminescence. In the decade following its development, the NanoLuc® toolbox has expanded to include NanoBiT® complementation, NanoBRET™ energy transfer methods, and new reagents such as the Nano-Glo® Fluorofurimazine In Vivo Substrate (FFz) which was designed for in vivo detection of NanoLuc® Luciferase, NanoLuc® fusion proteins or reconstituted NanoBiT® Luciferase. In addition to the aqueous-soluble reagents increased substrate bioavailability in vivo, with fluorofurimazine, NanoLuc® and firefly luciferase can be used together in dual-luciferase molecular imaging studies.

Here we spotlight some recent research that demonstrates how the expanded NanoLuc® toolbox can be adapted to use in non-mammalian models, shedding new light on fundamental biological processes and advancing our understanding of complex mechanisms in these diverse organisms.

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Clovibactin: A Revolutionary Antibiotic with No Resistance

Posted on by Riley Bell
Pills

Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections. It’s a leading mortality factor worldwide, but the newly discovered antibiotic, clovibactin, may offer a pivotal solution. It effectively kills drug-resistant bacterial pathogens without detectable resistance—even multidrug-resistant “superbugs.”

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RAF Inhibitors: Quantifying Drug-Target Occupancy at Active RAS-RAF Complexes in Live Cells

Posted on by Ken Doyle

Mitogen-activated protein kinases (MAPKs) are a large family of proteins that regulate diverse cellular functions in eukaryotes, including gene expression, proliferation, differentiation and apoptosis (1). MAPK signaling pathways typically include three sequentially activated kinases, and these pathways are triggered in response to extracellular stimuli, such as cytokines, mitogens, growth factors and oxidative stress (1). Ultimately, the signal is transmitted to the nucleus, with the activation of a specific transcription factor that modulates the expression of one or more genes.

Among MAPK pathways, the RAS-RAF-MEK-ERK signaling pathway has been studied extensively. Mutations in RAS family proteins and resultant dysregulation of the signaling pathway are implicated in a variety of cancers. Therefore, this pathway is a popular target for anticancer drug development.

An overview of the RAS-RAF-MEK-ERK signaling pathway.
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Ancient Worm Reveals Simple Hack for Remaining Ageless After 46,000 Years

Posted on by AnnaKay Kruger

If you could, would you enter a suspended metabolic state for the chance to reawaken 46,000 years from now, as you are today? For one nematode discovered in Siberian permafrost, the answer is a resounding “yes”. A study published in late July of this year details recent research that expands on a paper published in 2018 wherein scientists announced that they successfully reanimated a small but resilient nematode, or roundworm, who remained alive for tens of thousands of years in a state called cryptobiosis after being frozen in extreme Arctic soil conditions.

Blue roundworm on a black background
Caenorhabditis elegans, a type of roundworm whose dauer larvae are capable of cryptobiosis
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Breathtaking Breakthrough: How Gut Microbial VOCs Are Revealing Biomarkers, One Exhale At A Time

Posted on by Natalie Larsen

The human microbiome, the bustling cooperative of all the microscopic creatures that naturally colonize in and on our bodies, wields a surprising amount of influence over many of the unseen processes that are critical to our overall health and wellness. Over the course of decades, we have learned that this is particularly true for the microbes that reside in our gastrointestinal tract, collectively known as our gut microbiota.

Our gut microbiota is constantly communicating with our bodies, though our relationship with our gut can feel like trying to have a conversation with someone who only speaks a language we do not know or understand—you can take an educated guess at what they are saying based on their expressions and gestures, but the true message and meaning behind their actions is not always discernable. So while we can feel that someone in our gut is unhappy when we have a tummy ache, the true mechanism behind exactly who is unhappy and why, is not as obviously deduced or understood.

What if there was a tool that could help us more easily interpret the language of our microbiota, giving us the means to both better understand our microbiomes as well as to detect biomarkers of various diseases? Recent studies have shown that such a solution may be (quite literally) right under our noses: our breath.

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From Hit to Live-Cell Target Engagement Assay: DNA-Encoded Library Screens and NanoBRET Dye

Posted on by Tian Yang

Monitoring and quantifying drug-target binding in a live-cell setting is important to bridging the gap between in vitro assay results and the phenotypic outcome, and therefore represents a crucial step in target validation and drug development (1). The NanoBRET™ Target Engagement (TE) assay is a biophysical technique that enables quantitative assessment of small molecule-target protein binding in live cells. This live-cell target engagement assay uses the bioluminescence resonance energy transfer (BRET) from a NanoLuc® luciferase-tagged target protein and a cell-permeable fluorescent tracer that reversibly binds the target protein of interest. In the presence of unlabeled test compound that engages the target protein, the tracer is displaced, and a loss of BRET signal is observed. Due to the tight distance constraints for BRET, the signal measured is specific to the target fused to NanoLuc® luciferase.

Live-cell target engagement assay using NanoBRET to measure small molecule binding to a target transmembrane protein.

Promega offers over 400 ready-to-use assays for multiple target classes, including kinases, E3 ligases, RAS, and many others. For targets that do not have an existing NanoBRET™ TE assay, Promega offers NanoBRET™ dyes, NanoLuc® cloning vectors, and NanoBRET™ detection reagents to develop novel NanoBRET™ TE assays.

To learn more about the NanoBRET™ TE platform, see the NanoBRET™ Target Engagement Technology Page on our website.

One critical component in the development of novel NanoBRET™ TE assay is the creation of the cell-permeable fluorescent tracers (NanoBRET™ tracers) against the target protein of interest. The tracers are bifunctional, consisting of a NanoBRET™-compatible fluorophore and a target-binding moiety connected by a linker. While the NanoBRET™ 590 dyes have demonstrated consistently robust cell permeability and optimal spectral overlap with NanoLuc® for BRET, a ligand capable of binding to the target protein of interest needs to be identified to generate a NanoBRET™ tracer.

What Are DNA-Encoded Libraries?

DNA-Encoded Libraries, (DELs), have emerged as powerful tools for discovering small molecule ligands to target proteins of interest at an unprecedented scale. . owing to the ability of a DEL  to enable the synthesis of larger libraries of compounds and to target proteins without any prior structural knowledge of the proteins or their ligands (2). Because each member of a DEL contains a DNA barcode and a small molecule separated by a linker, DEL is primed for discovering leads within therapeutic modalities that rely on bifunctional chemistry, such as proteolysis targeting chimeras (PROTACs). Since NanoBRET™ tracers are also bifunctional, ligands identified from DEL selections could serve as ideal candidates for developing novel NanoBRET™ tracers that can enable NanoBRET™ TE assays for new targets.

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Cancer Preventing Vaccines: Unleashing the Potential of Tumor Antigens

Posted on by Sara Millevolte

It has been more than 100 years since Dr. William B. Coley, known today as the “Father of Immunotherapy,” made the first recorded attempt to mobilize the immune system as a means of treating cancer (9). Decades later, the discovery of T cells and the vital role they play in the immune system set the groundwork for many new immunotherapy treatments, such as those involving monoclonal antibodies, cytokines, CAR T cells, and checkpoint inhibitors.

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