Highly Visible Invisible Food Safety

  • Recent FDA Food Safety Act Focuses on Prevention Rather Than Response
  • PCR Provides a Powerful Approach for Assuring Foodborne Safety
  • Safer Food via Immuno-PCR Commercialized by Invisible Sentinel 

Annual Cost of Foodborne Illness and U.S. FDA Response

Every year contaminated food sickens some 48 million people in the U.S., necessitates 128,000 hospitalizations, and results in 3,000 deaths, according to recent estimates from U.S. Centers for Disease Control and Prevention. Extrapolating these numbers to other developed countries isn’t straightforward, but I used available information to guess that the number of cases of foodborne illness worldwide is roughly 4-5 times greater than the US.

In addition to the toll in human suffering, food contamination that is discovered too late exacts a heavy financial cost on the food industry and the public. A study supported by the Pew Charitable Trusts has estimated that food contamination costs the United States about $152 billion a year after accounting for lost workdays and reduced quality of life, as well as medical expenses.

Continuing outbreaks of foodborne illness have led consumer groups to call for tighter regulation. The result was the FDA Food Safety Modernization Act (FSMA)—the most sweeping reform of U.S. food safety laws in more than 70 years—signed into law by President Obama on January 4, 2011. It aims to ensure the U.S. food supply is safe by shifting the focus from responding to contamination to preventing it. Here’s how.

The U.S. FDA now has a mandate to require food-safety controls. Companies across the food-production and food-distribution network must write outbreak-prevention plans, monitor the performance of their controls, and specify the corrective actions they will take when necessary.

A detailed summary of the FSMA and a host of related information can be read about at this link that includes Guidance for Industry, Final Rules, and Presentations.

PCR Powered Prevention 

In a nutshell, the well-known power of PCR to unambiguously identify and quantify microorganisms lends itself to prevention of foodborne contamination from entering the food supply. Other important attributes of PCR for such prevention are its highly evolved instrumentation and ancillary sample preparation methods, which together provide for fast time-to-results in either centralized labs that receive express-shipped samples, or decentralized (aka point-of-need) facilities at or very near the food source.

Most major commercial suppliers of PCR instrumentation and reagents offer a line of products aimed at food safety per se or quality control for microorganisms that are detrimental to taste, smell, shelf life, and other producer or consumer concerns. An example of such is ETS Labs in California, which uses real-time PCR to detect a full range of wine and juice spoilage organisms to help ensure quality in the wine making industry. This genetic analysis method, which utilizes TriLink’s Hot Start CleanAmp dNTPs under a licensing agreement, detects microbial populations directly in wine or juice. Results are routinely reported within two business days, giving winemakers the ability to address problems before wine defects occur.

Regular readers of this blog will recall that I have touted the advantages of PCR in various contexts, including some aspects of food chain validation and tracing from “farm-to-fork.” However, as indicated above, the present post is focused on preventing foodborne illnesses and showcasing innovators in this space, namely Invisible Sentinel.

Highly-Visible Invisible Sentinel

Truth be told, I can’t recall how I first came across Invisible Sentinel, but I’m glad I did because it’s an interesting story from the perspective of innovation in food safety technology as well as opportunity in an emerging market.

Taken from phillymag .com

Technology-wise, the first thing that caught my attention was the remarkably small size of the PCR read-out device developed by Invisible Sentinel, which is pictured below. Before getting into the specifics of what’s packed inside of this tiny gizmo, I should mention that there are up-front sample prep and PCR thermal cycling steps that must be performed before using the device. These steps have been simplified but involve conventional approaches that can be read about at this link rather than discussed here.

Much more intriguing to me was how PCR amplicons are detected by Invisible Sentinel’s Veriflow® DNA Signature Capturing Technology, which more accurately involves visualization by eye as opposed to fluorescent signal detection commonly used for real-time PCR measurements. Since functional details for this visualization system are not provided on Invisible Sentinel’s website or various YouTube videos, I’ll briefly summarize below what I found in a recently issued Invisible Sentinel patent that describes its version of what is known as Immuno-PCR.

The following schematic, taken from this patent, depicts visualization of two different amplicons derived from 5’-labeled primers: digoxigenin/TAMRA amplicon 20 and FITC/TAMRA amplicon 40 using three antibodies 15, 30, and 50 that are either attached to a membrane, bridge, or bind streptavidin-gold nanoparticles. The latter nanoparticles are visualized by eye, but only if both amplicons form the complex shown. Variations of this scheme can be used for visualization of a single amplicon.

Taken from US Pat. No. 9,347,938

Visualization in this manner is formally analogous to pregnancy strip tests that show two bands for a positive result and only one band for a negative result. Interested readers should consult the aforementioned patent for details regarding how input amplicons undergo lateral flow to ultimately bind to antibody 10 attached to the test membrane shown above.

Exemplary Applications

According to Invisible Sentinel, 114 companies in the U.S. and more than 50 internationally use the technology at more than 250 different sites in 18 countries.

For example, Wawa Inc. (which owns dairy and beverage manufacturing plants as well as 715 convenience stores in six states) has adopted Veriflow®, as has Refresco Gerber Partner for monitoring juice spoilage. WholeVine Products in California, which produces a variety of products from grape seeds and skins, has begun using Veriflow® to make sure its plant equipment and surfaces are pathogen-free.

Although Invisible Sentinel’s website provides a list of currently available tests, I thought it would be useful to provide the following links to several self-explanatory published applications that I found by searching Google Scholar:

Invisible Sentinel Identifies New Market Opportunities for PCR

Invisible Sentinel was started by a pair of entrepreneurs with science backgrounds. Nicholas Siciliano, 37, graduated from Villanova with a degree in chemistry in 2004 and obtained a doctorate in immunology and microbial pathogenesis from Thomas Jefferson University in 2015. In between, he was a biotech consultant and worked as a researcher at the University of Pennsylvania School of Medicine.

Nicholas Siciliano (left) and Benjamin Pascal in their lab. Taken from articles.philly .com

Benjamin Pascal, 35, has a bachelor’s degree in political communication from George Washington University in 2003 and a master’s in business administration from Lehigh University in 2009. He learned biology at the National Institute for Medical Research in London, and then spent several years in R&D at B. Braun Medical Inc.

According to an article in the NY Times, the two wanted to create a diagnostic device that was faster, easier and cheaper to use. They began with $235,000 from friends and family, but the recession made it tough to bring institutional investors onboard. In 2009, they raised another $1.1 million from friends and family, $2 million more in 2011, and raised $7 million at the end of 2013.

Invisible Sentinel’s sales have been on the rise. The company posted revenue of $50,000 in its first year of sales in 2013, $1.1 million in 2014 and more than $4 million in 2015. It has ambitious projections of $30 million in 2018 and $60 million in 2020. The company expected to turn a profit in 2016.

While Invisible Sentinel may have been one of the first to identify the significant market opportunity for food safety monitoring devices, they currently face formidable competition in larger companies such as Romer Labs’ RapidChek®, Bio-Rad Laboratories’ iQ-Check® and DuPont’s Bax® system. Invisible Sentinel is hoping to capture significant market share with its low cost of entry and easy-to-use system. The company can reportedly set up an in-house lab for about $5,000 and train almost anyone to use it in less than a day. Invisible Sentinel kits cost more than others (about $10 per test compared to an industry average of $4 to $8), but the lower capital equipment and lab set up costs are said to greatly offset the higher test costs.

In closing, I hope that you have found this piece on food safety and immune-PCR in the context of Invisible Sentinel to be a nice example of how nucleic acids-based technology is enabling improved food safety.

As usual, you are welcomed to share your comments here.

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Ocean ‘Dandruff’ DNA to Better Study Marine Biology

  • DNA Barcoding for all Organisms has Numerous Applications
  • DNA Barcodes from Water Samples Greatly Aide Marine Biologists
  • Aquatic Environmental DNA (eDNA) Proves to be Informative ‘Dandruff’

Human DNA identity analysis is now commonplace methodology that’s frequently featured in newspaper stories, TV crime series, or “who dun it” movies. The same principle (i.e. using a characteristic DNA pattern or signature) applies to identification of all animals, birds, insects, and microbes. Actually, DNA barcoding extends to any organism, whether it is alive or has been dead for hundreds of thousands of years (so long as it’s preserved by fossilization).

Taken from gajitz.com

Marine biologists face a serious challenge with accounting for very diverse forms of marine life that exists in a mindboggling huge volume of water. Consequently, it’s not surprising that analysis of water-borne, marine DNA barcodes—as proxies for going to and counting fish—is rapidly trending in utility and importance. Known formally as environmental DNA (eDNA), the aquatic version has been humorously referred to as ocean ‘dandruff’ by Christopher Jerde of the University of Nevada in Reno (which, ironically, is landlocked and distant from any ocean.) But I digress. Before diving further (pun intended) into ocean dandruff, let’s briefly review the background of DNA barcoding.

DNA Barcodes 101

Prof. Paul Herbert. Taken from uoguelph.ca

In 2003, Prof. Paul Herbert and coworkers in the Department of Zoology at the University of Guelph in Canada published a seminal study titled Barcoding animal life: cytochrome c oxidase subunit 1 (CO1) divergences among closely related species that fundamentally changed the field of taxonomy. In a nutshell, Herbert’s team showed it was feasible to classify millions of species based only on DNA sequence of the mitochondrial gene CO1. In the intervening, relatively short amount of time, there have been thousands of publications dealing with applications and extensions of this concept, which is now recognized to be very powerful and promising albeit with some limitations.

Typically, DNA barcodes are identified by sequencing after PCR amplification of one or more specific genetic loci such as CO1. Following proof that a DNA barcode can differentiate the species of interest, single- or multiplex quantitative PCR (qPCR) can be used to enumerate relative amounts of sample from the field.

The advent of high-throughput sequencing technologies applicable to complex mixtures of individually tagged samples then gave rise to “metabarcoding,” about which interested readers can consult many publications for specific topics.

Craig Venter steers his research yacht, Sorcerer II, under the Sydney Harbour Bridge in his quest to collect microbes from the world’s waters. Photo: Dallas Kilponen. Taken from smh.com.au

BTW, among the many pioneering scientific ventures by uber-famous Craig Venter, is his Global Ocean Sampling Expedition aboard his research yacht, Sorcerer II. The expedition is a quest to unlock the secrets of the oceans by sampling, sequencing and metabarcoding DNA of all (or most) microorganisms living in these waters.

Lest you think this was a well-intended but unproductive journey—some say junket—by Venter and coworkers, here’s a link to peruse 16 resultant publications that I found by searching PubMed. To watch and listen to Venter talk about this work, you can click here for an educational and entertaining—as usual with Venter—TED Talk on Sampling the Ocean’s DNA that’s had over 550,000 views!

Ocean ‘Dandruff’

Now that we’ve covered the basics of DNA barcoding and metabarcoding, let’s turn back to ocean dandruff. Dandruff, simply put, is dead skin cells. Using dandruff as an intended witty metaphor for ocean eDNA is a bit misleading as marine eDNA is comprised of a complex mixture of cellular matter from scales, feces, decomposing tissue, etc. of fish and all other present or past sea creatures. Consequently, the design and specificity of primers for PCR is of paramount importance for obtaining—let alone interpreting—DNA barcodes based on fragment size or sequence.

As reported by Miya et al., monitoring the occurrence of fish species-specific eDNA PCR fragments (~70–300 bp) has traditionally used conventional electrophoretic gel separation and detection. More recently, qPCR using fluorogenic probes has been employed owing to the method’s sensitivity, specificity and potential to quantify the target DNA. For example, it has been possible to accurately estimate the biomass of common carp in a natural freshwater lagoon using qPCR of eDNA concentrations and biomass in aquaria and experimental ponds.

Miya et al. also describe the development of a set of PCR primers for metabarcoding mitochondrial DNA of 880 species of fish. They sampled eDNA from four tanks with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing. Out of the 180 marine fish species contained in the four tanks, they detected 168 species (93.3%) distributed across 59 families and 123 genera. That’s quite an impressive accomplishment.

Ocean Dandruff Case Studies

Since there are so many fish-related applications of DNA barcodes, I’ve selected several recent examples that are indicative of the utility of ocean ‘dandruff’—and are quite interesting, in my opinion. The first case in point exemplifies how eDNA can be used to deal with rare and endangered species, which are either very hard to find or can be dangerously distressed by catching to obtain samples.

Green SturgeonBergman et al. report that a decline in abundance of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning by these Green Sturgeon are effective at monitoring fish densities in concentrated pool habitats, results do not scale well—pun intended. By contrast, eDNA provides a relatively quick, inexpensive tool to efficiently identify and monitor Green Sturgeon DNA.

Taken from mthsecology.wikispaces.com

These investigators concluded that follow-on work based on this first-ever eDNA study of Green Sturgeon has the potential to provide better knowledge of the spatial extent of Green Sturgeon spawning that could help identify previously unknown spawning habitats and discover factors influencing habitat usage, guiding future conservation efforts.

Monterey Bay—The second case study, by Port et al., involves taking stock of the marine mammals and fish in Monterey Bay using eDNA and, importantly, comparing the results obtained to those from traditional dive surveys.

In brief, this team of researchers from several universities and the Monterey Bay Aquarium Research Institute found that eDNA assessments picked up almost all the organisms scuba divers spied underwater—plus many more that human eyes missed. Here’s some detail on how they did this.

At each scuba survey location as well as at sites offshore, ~1 gallon of water was sampled several feet above the bottom. Four types of habitats were sampled: sea grass beds, Monterey Bay’s unique “Kelp Forest,” sandy areas and rocky reefs. Onshore, in a “clean” (DNA-free) lab, these water samples were filtered to collect cells containing eDNA for storage at −80 °C until eDNA extraction at a university clean lab. A vertebrate‐specific primer set targeting a small region of the mitochondrial DNA 12S rRNA gene was used for PCR followed by gel purification.

Researchers collecting water in Monterey Bay for eDNA analysis. Courtesy Jesse Port. Taken from mercurynews.com

After quantification, pooled amplicons (each having a sample index sequence) were paired-end sequenced on the Illumina MiSeq platform using a 20% PhiX spike‐in control to improve the quality of low‐diversity samples. The conclusions are worth quoting because—in my opinion—the findings represent a new era in marine biology based on nucleic acid analysis:

“We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 meters. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.”

Restated more simply, eDNA analysis of the water picked up 11 of the 12 fish and marine mammals that the divers observed, and—importantly—identified 18 additional animals the divers missed! The efficiency and improvement offered by eDNA analysis compared to traditional seek-and-count methods has been echoed in an editorial I found by Hoffmann et al. titled, tongue-in-cheek, Aquatic biodiversity assessment for the lazy.

Invasive Gobies—The third and final case study deals with detection of invasive, non-native fish to assess whether eDNA can provide a better advanced warning system for detecting these unwanted creatures and implementing eradication steps.

Gobies are an invasive fish species that has colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby (Neogobius melanostomus), pictured below, is among the worst invaders in Europe. Current methods to detect the presence of these gobies are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile.

Taken from animal.memozee.com

To improve monitoring, Swiss and Canadian collaborators developed an assay based on the detection of eDNA in river water, without detecting any native fish species, which is obviously an important assay criterion. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by novices and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s reports and fish community monitoring.

I could go on and on with examples of utility and the many advantages provided by eDNA for marine biology, but I’m sure you get the picture. I hope that you agree with me that eDNA analysis is a very valuable type of trending nucleic acid-based methodology.

As usual, your thoughts or comments are welcomed.

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Studying Telomeres in Space

  • Telomeres are DNA Biomarkers for Your Biological “Age”
  • Telomere Shortening Due to Stress was Expected During Spaceflight, but Exactly the Opposite has Been Found
  • Raising New Questions to Answer

After you read this blog about studying telomeres in space, I think you will agree with my opinion that scientific advances can sometimes occur amazingly fast. Telomeres (which are peculiar DNA structures that I’ll explain below) went from esoteric Nobel Prize subject matter in 2009 to the focus of spaceflight science in just six short years. Now, telomeres are being investigated by PCR on the International Space Station (ISS)! With a wink and a nod to Star Trek, this is indeed “warp speed” progress!

Taken from keywordsuggest.com

What are telomeres?

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates like us, the repetitive sequence of nucleotides in telomeres is TTAGGG, with the reverse complementary DNA strand being AATCCC, as depicted below. This sequence of TTAGGG is repeated ~2,500 times in humans.

During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, causing the end of the chromosome to be shortened in each replication. The telomeres are thus disposable “buffers” at the ends of chromosomes which are truncated during cell division, as depicted below.

Taken from weeklyglobalresearch.wordpress.com

Telomers, however, are replenished by an enzyme named telomerase. This peculiar enzyme has an embedded RNA template and incorporates DNA nucleotides, as depicted below, and is therefore a special kind of reverse transcriptase. In people, it has been found that telomeres shorten with age in all replicating somatic cells that have been examined. In fact, average telomere length declines from about 11 kilobases at birth to less than 4 kilobases in old age, with the average rate of decline being greater in men than in women. Thus, telomere length can serve as a biomarker of a cell’s biological (versus chronological) “age” or potential for further cell division.

Taken from 2014hs.igem.org

‘Houston, we have a problem’

This now famous phrase, which was used in the past tense by the crew of the Apollo 13 moon flight to report a major technical problem back to their Houston base, echoed in mind when I learned that space flight might lead to telomere shortening. Yikes! This molecular-level change could indeed be a serious problem, and was first suggested by findings from laboratory microgravity simulations reported in 2008 by Chinese researchers. Since it was known that space flight leads to bone loss, they cultured bone stem cells (BSCs) under simulated microgravity in a rotary cell culture system.

This led to significantly decreased activity of telomerase. It was postulated that reduced bone formation in space flight may partly be due to the altered potential differentiation of BSCs associated with telomerase activity, which plays a key role in regulating the lifespan of cell proliferation and differentiation. Additionally, telomerase activation or telomerase replacement may act as a potential countermeasure for microgravity-induced bone loss.

Taken from energeticnutrition.com

If you’re thinking that these “potential countermeasures” are fanciful, you’d better think again. I recently came across a patent that was published last year on methods and compositions for increasing telomerase activity in cells, including pharmaceutical formulations. Moreover, there are now various commercially available supplements claiming to promote telomerase activity, such as that picture below. I hasten to add that I do not advocate use of any such supplement, and that interested readers should consult their primary care physician or certified nutritionist.

Twins and telomeres

Although identical twins are almost the same genetically, differences in environment, diet and other outside factors can affect their health in different ways. Consequently, identical twins have been enrolled in various studies that require deciphering effects due to “nature vs. nurture” (i.e. intrinsic genetics vs. external factors). Part of the Twins Study supported by NASA was aimed at examining the effects of space travel on one of a pair of twins: astronaut Scott Kelly, who stayed on the ISS for one year, while his twin brother, Mark, remained on Earth. In brief, Prof. Susan Bailey at Colorado State University is exploring differences between the twins’ telomeres to determine if telomeres respond differently to spaceflight and then how such changes relate to the various medical endpoints studied by other Twins Study investigators.

Scott Kelly (left) and his identical twin brother Mark in 2015 prior to Scott’s one-year mission to the ISS. Taken from space.com

Preliminary research results for this part of the NASA Twins Study (reported at NASA’s annual Investigators’ Workshop earlier this year) were a quite surprising because they were opposite of what was expected, thus raising more questions than providing answers. It had been theorized that exposure to microgravity and stress during prolonged spaceflight would shorten telomeres, but instead Bailey’s team found telomeres in Scott’s white blood cells increased in length while in space! This finding was rationalized as being due to increased exercise and reduced caloric intake during the space mission. Upon his return to Earth, however, these telomeres began to shorten again.

This is yet another example of a biomedical phenomenon being far more complex that first theorized, and one that becomes less understood as more and more data are obtained. We’ll all have to patiently stay tuned for how this telomeres-in-space story evolves. The good news, as I’ll explore in the next and final section of this blog, is that there are exciting plans to use PCR to measure telomere length extraterrestrially! This is very “far out” science—pun intended.

Studying telomeres in space by PCR

Taken from geeky-gadgets.com

The aforementioned Twins Study involved taking blood samples from an astronaut during spaceflight for lab analysis upon return to Earth. To obtain much more data, and to do so in real time while in space, NASA launched the Genes in Space-2 mission in April 2017. The goal is to determine whether astronauts aboard the ISS can analyze telomeres by PCR reactions in a small thermal cycling device (miniPCR system) and thus measure and monitor telomere changes during spaceflight.

In addition to testing the miniPCR system, the Genes in Space-2 mission has a secondary goal to test the feasibility of techniques used to measure telomere length. Currently, Single Telomere Length Analysis (STELA) is the only suitable technique for use on the ISS due to technical requirements. The Genes in Space-2 mission will also be testing the feasibility of a loop-mediated isothermal amplification (LAMP) colorimetric assay for detection of amplification aboard the ISS. Please stay tuned for updates on the outcome of these very important feasibility experiments.

Scheme for STELA procedure. Taken from Xing et al. (2009)

Julian Rubenfein. Taken from nydailynews.com

As a side note, the Genes in Space competition for 2017 selected this experiment on telomere amplification in microgravity from 375 submissions by nearly 850 students in grades 7 to 12 from across the US. This telomere experiment was proposed by 17-year old Julian Rubinfien from Stuyvesant High School in New York City, who is pictured below. I encourage you to read this interesting, although lengthy interview about his background and the experimental rationale. What’s even more interesting is this short video of Julian at the launch and his comments—very impressive!

I strongly encourage you to read more about all the award-winning experiments in this exciting round of competition among young, highly motivated, advanced students, who I’m sure will be successful in whatever they do in the future.

Your thoughts or comments here are welcomed.

Postscript

Profs. Elizabeth Blackburn and Carol Greiner—who received a Nobel Prize in 2009 for seminal work on telomeres—co-founded Telome Health Inc. (THI) in 2010 to leverage the predictive power of telomere-length assays to help assess health status, disease and mortality risk, and response to specific therapies. THI subsequently announced TeloTest™ as a diagnostic test that measures average telomere length by qPCR. TeloTest™ was the first saliva-based telomere test available on the market, and is currently offered by a company named TeloYears.

The clinical utility of testing telomere length in a saliva-based test was recently reported from an independent, large clinical study sponsored jointly by Kaiser Permanente, University of California, San Francisco (UCSF), and National Institutes of Health. In the study, the average telomere length of 100,000 Kaiser patients was measured and analyzed relative to other health domains and clinical outcomes.

My recently obtained TeloTest™ results from TeloYears indicated that my biological age is 4 years older than my chronological age. Naturally, I was hoping to learn that my telomere-based age would be less than my actual age. Alas, the results are what they are, so I’ll be following diet, exercise, sleep, and stress-management recommendations you can read about at TeloYears Learning Center.

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DNA Day 2017

  • There are Now Millions of DNA-Related Publications
  • Some of the Top 5 Cited Papers on DNA Will Surprise You
  • You Probably Won’t Guess Top 5 Most Frequently Cited

Deciding what to post here in recognition of DNA Day 2017 was just as challenging as it has been in past years, primarily because there’s so many different perspectives from which to choose. After much mulling, and several abandoned approaches, I settled on featuring DNA publications that have received the most citations, as an objective metric—not just my subjective opinions about topics I think are significant or otherwise interesting.

Before getting to the numbers of DNA-related papers and some of the most cited papers, here’s a quick recap of what was posted here in the past, starting with the inaugural blog four years ago:

2013—60th Anniversary of the Discovery of DNA’s Double Helix Structure

2014—My Top 3 “Likes” for DNA Day

2015—Celebrating Click Chemistry in Honor of DNA Day

2016—DNA Dreams Do Come True!

Explosive Growth of DNA Publications

Regular readers of my blogs will know that I frequently use the NIH PubMed database of scientific articles to find publications by searching keywords, phrases, or authors. A convenient feature of these searches is providing “results per year” that can be exported into Excel for various purposes. Some preliminary searches indicated that DNA-related articles can be indexed by either DNA or PCR, or cloning, or other terms among which sequencing was notable. The majority, however, were indexed as either DNA or PCR, which together gave nearly 1.7 million items—an astounding number. This number is even much greater since PubMed excludes some important chemistry journals, as well as patents.

Diving deeper into these numbers, I thought it helpful to look at the publication volumes and rates for DNA, sequencing DNA, and PCR through 2015 starting from 1953, 1977, and 1986, respectively. These respective dates correspond to seminar publications by Watson & Crick, Maxam & Gilbert, and Mullis & coworkers. The results shown in the following graph attest to my often stated “power of PCR” as premier method in nucleic acid research, which we’ll see again below in another numerical context.

Top 5 Cited Papers

During my perusal of the above literature in PubMed generally related to DNA, I thought it would be interesting to find, and share here, which specific papers have the distinction of being most frequently cited. Citations are not available in PubMed, but are compiled in Google Scholar, which led me to these Top 5 that are listed from first to fifth.

Frederick Sanger (1918-2013) Taken from newscientist.com

  1. DNA sequencing with chain-terminating inhibitors

Frederick Sanger, the eponymous father of the “Sanger sequencing” method published in 1977, received the 1980 Nobel Prize in chemistry for this contribution. He also received the 1958 Nobel Prize in chemistry for sequencing insulin, and is the only person to win two Nobel Prizes in chemistry. Uber-famous DNA expert Craig Venter is quoted as saying that ‘Fred Sanger was one of the most important scientists of the 20th century,’ [who] ‘twice changed the direction of the scientific world.’

  1. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method

Kenneth J. Livak, PhD
Taken from archive.sciencewatch.com

The most commonly used method to analyze data from real-time, quantitative PCR (RT-qPCR) experiments is relative quantification, which relates the PCR signal of the transcript of interest to that of a control sample such as an

untreated control. The derivation, assumptions, and applications of this method were published in 2001 by Livak & Schmittgen. I overlapped with Ken Livak at Applied Biosystems, which pioneered commercilaization of RT-qPCR reagents and instrumentation at the time. He is currently Senior Scientific Fellow at Fluidigm Corp.

Sir Edwin M. Southern Taken from ogt.co.uk

3. Detection of specific sequences among DNA fragments separated by gel electrophoresis

Sir Edwin Mellor Southern, FRS, the eponymous father of “Southern blotting” DNA fragments from agarose gels to cellulose nitrate filters published in 1975, is a Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College. He is also Founder and Chief Scientific Advisor of Oxford Gene Technology.

  1. Prof. Bert Vogelstein, MD
    Taken from hhmi.org

    A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

This paper by Feinberg & Vogelstein published in 1983 describes how to conveniently radiolabel DNA restriction endonuclease fragments to high specific activity using the large fragment of DNA polymerase I and random oligonucleotides as primers. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments. His group pioneered the idea that somatic mutations represent uniquely specific biomarkers for cancer patients, leading to the first FDA-approved DNA mutation-based screening tests, and now “liquid biopsies” that evaluate blood samples to obtain information about underlying tumors and their responses to therapy (an area that I’ve touted in previous blogs). A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 109 dpm/μg of DNA can be obtained using relatively small amounts of precursor. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments.

  1. Kary B. Mullis, PHD
    Taken from TED.com

    Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

In 1988, Kary B. Mullis and coworkers (then at Cetus Corp.) published in venerable Science a method using oligonucleotide primers and thermostable DNA polymerase from Thermus aquaticus to amplify genomic DNA segments up to 2000 base pairs to detect a target DNA molecule present only once in a sample of 105 cells. Since that time, polymerase chain reaction (PCR)-related technology has evolved to now routinely enable a variety of single-cell analyses of DNA or RNA. Dr. Mullis received the 1993 Nobel Prize in chemistry for his 1983 invention of PCR, which his website says ‘is hailed as one of the monumental scientific techniques of the twentieth century.’

Top 5 Papers by Citation Frequency

While writing the above section, it occurred to me that ranking these five publications by total number of citations-to-date in Google Scholar doesn’t account for differences in the number of years between the year of publication and now. I did the math to calculate the average citation frequency per year, and here’s the totally surprising—to me—result: relative gene expression methodology published by Livak & Schmittgen is by far the most frequently cited of the Top 5, according to this way of ranking:

  1. 2001, relative gene expression, Cited by 69560 = 4,637 avg. citations per year
  2. 1977, Sanger sequencing, Cited by 32662 = 1,701
  3. 1975, Southern blotting, Cited by 21201 = 796
  4. 1988, PCR, Cited by 18785 = 671
  5. 1983, oligolabeled DNA, Cited by 21200 = 642

I should point out that, as transformative methods such as these gradually become widely recognized as “standard procedures,” researchers tend to feel it unnecessary to include a reference to the orignal publication. Consequenly, citation frequency decreases with time even though cummulative usage increases. In other words, 25 years from now average citations per year for relative gene expression will have likely decreased, and be surpassed by a new “method of the decade,” so to speak.

Prediction for the Future

This line of reasoning leads me to close with some speculation about what DNA-related technique might emerge as the next “method of the decade” that tops the above ranking by citation frequency.

My guess is that it will be Multiplex genome engineering using CRISPR/Cas systems by Zhang & coworkers that has been cited by 4145 at the time I’m writing this piece, only four years from its publication in venerable Science in 2013. Some of my blogs have already commented on various aspects of CRISPR/Cas9, which is among genome editing tools offered by TriLink.

As usual, your comments are welcomed.

Finding Frankfurter Fraud Featuring Famously Familiar PCR

  • While Thousands of PCR-based Tests for Food Authentication Exist, Commercial Adoption is Lax
  • PCR Tests for Halal Frankfurter Products Reported by Malaysian Team
  • PCR-enabled Next Generation Sequencing of USA Frankfurters Exposes Extensive Mislabeling and Adulteration

Regular readers of this blog will know that (a) I favor alliterations, (b) frequently feature PCR-based topics, and (c) I am fond of food facts involving nucleic acids, all of which are found in the title of this posting. While writing style and food are both a matter of taste, so to speak, it’s almost impossible to comment on nucleic acids without involving PCR, in one way or another, as PCR is—in my opinion—the most widely used and important method in molecular biology.

Having said this, and knowing that this summer alone Americans will likely consume an estimated 7 billion (!) hot dogs, (a.k.a. frankfurters or wieners), I thought it apropos to now feature finding frankfurter fraud by PCR. But before getting to that, I thought it’s worth commenting first on a frankfurter vs. wiener vs. hot dog and other “meaty” definitions to get us “linked” up—please pardon the puns, another of my penchants.

Frankfurter, Wiener, Hot Dog Lexicon

Frankfurter sausages and sauerkraut. Taken from tripadvisor.com

While it’s a fact that a resident of Frankfurt, Germany is properly called a Frankfurter, one of Frankfurt’s pork sausage specialties is also called a frankfurter—but spelled with lower case f—and is short for Frankfurter Würstchen, which go back to the 13th century. By the same token, wiener refers to a pork and beef sausage specialty introduced in the 18th century in Vienna, Austria—a city called Wien in German, and hence the word wiener.

Frankfurters and wieners look very similar, with the main culinary distinction being absence of serving with the bun, which by contrast is characteristic of a hot dog. Readers interested in the origin of the hot dog’s name and bun usage will learn at this link—pun intended—that there are various and widely different claims for this name and usage.

Hot dogs in buns. Taken from clowns4kids.com

Dog Factory, a short film by uber-famous Thomas Edison in 1904 poked fun at what went into hot dogs. Taken from wikipedia.org

In one such claim, the term dog is said to have been linked—there I go again—to sausages made from dog meat, as popularized in an old spoof by Thomas Edison (!) pictured below. This issue of what meat(s) hot dogs and similar sausages contain now segues into finding frankfurter fraud using PCR.

Finding Food Fraud by PCR

Before getting to the meat of this matter (oops!) involving frankfurters, I thought it would be informative to provide some larger perspective on finding food fraud, generally, utilizing the power of PCR for specific detection of nucleic acids that are characteristic of a given species of any food whether it be meat, fish, vegetable, etc. My search of Google Scholar for articles with all of the terms “food, fraud, and PCR” gave ~3,900 items. Here are some selected samplings, the first of which was taken from several I found that used TriLink products—hooray!

  • US Food & Drug Administration researchers employed species-specific primers from TriLink for multiplex real-time PCR analysis of salmon and trout species in a range of 80 commercial products in North America. 4 instances (5%) of fraud were found.
  • By contrast, a whopping 40% of commercial pet food products tested by PCR for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) were found to be potentially mislabeled, according to a study by academic researchers.
  • Saffron produced from dried stigma of Crocus sativus is considered to be the most expensive food spice in the world, as ~200,000 (!) flowers must be carefully hand-picked (!) to produce only 1 kg of spice. Combating saffron fraud with PCR has already led to ~150 publications (!).
  • PCR-based food authentication to screen for possible allergens or GMOs is important for prevention of potentially life-threatening food contamination or alleviating consumer perceptions—or perhaps misperceptions, as I’ve commented on previously.

Finding Frankfurter Fraud by PCR

As for finding frankfurter fraud by PCR, the aforementioned ~3,900 items from my Google Search of food, fraud, and PCR was sub-searched for frankfurter, which led to only 3 reports titled as follows:

The third item, which is by a team of Malaysian researchers and is the most recent, offers some interesting introductory perspectives on strict religious, cultural, or geographical restrictions over the consumption of certain meats in the context of commercial frankfurters.

For example, pork is totally unacceptable to Malaysia’s large Muslim population, as well as Jewish and certain Christian denominations. On the other hand, Egyptians prefer buffalo because of their cultural preferences, while some Indians and Europeans avoid beef because of religious requirements and the fear of bovine spongiform encephalopathy (aka “Mad Cow Disease”—click here for an FDA update), respectively.

In this Malaysian study, 100% beef, buffalo, or pork frankfurters were prepared as models, and then fully heat-processed in the laboratory to simulate conventional manufacturing procedures. Additional beef, buffalo, or pork frankfurter models were deliberately contaminated by “spiking” in 1%, 0.5%, or 0.1% of buffalo and pork, beef and pork, and beef and buffalo meat, respectively. PCR was then performed using species-specific primer pairs for two genes (cytochrome b and NADH dehydrogenase subunit 5) for cross-validation. Twenty different halal-branded (i.e. pork-free) “beef” frankfurters from Malaysian markets were tested. While no pork was detected in any of the tested “beef” frankfurters, they were all beef- and buffalo-positive, thus revealing that all of the investigated Malaysian commercial “beef” products were buffalo-adulterated.

Halal Certification by PCR

The above mentioned concern for non-pork halal-assurance piqued my interest as to the extent of PCR usage for halal-related certification, and my subsequent Google Scholar search for “halal, certification, PCR” gave nearly 350 items. This relatively large number of reports signals quite widespread adoption of PCR. I encourage those interested to peruse these items later, but will mention here that the following article is the most cited—close to 100 citations as of February 2017—Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

In closing, I think it’s worth noting in the context of halal certification—and increasingly popular “democratization” of technology, about which I’ve previously offered comments—a French start-up company now offers an antibody-based “dip stick” kit (Halal Test) for anyone to use for pork-free halal-assurance at home or even when eating out. Amazing.

Taken with copy write permission from French duo launch HalalTest: ‘We want to democratize analysis’ by Rachel Arthur+Rachel ARTHUR, 05-Nov-2014

Hot Dog! — There’s Now an NGS Food Authentication Service Company

Frankly—pun-to-be intended—I don’t know how “hot dog!” became an exclamatory phrase for good news. It applies, however, to the fact that Clear Labs Inc. (a 2014 start-up in Palo Alto, California) has proven that several issues of concern for consumers of hot dogs can be successfully addressed by PCR/NGS methods.

In a Clear Labs’ poster abstract for the 2016 International Association of Food Protection meeting, results were reported for a study of 345 hot dog products sold by national brands to compare product label information and ingredient lists with the results of NGS analyses. Following DNA extraction, universally accepted regions for animals, plants, and bacteria were PCR-amplified for NGS, which revealed that ~15% of these products had ingredient substitution, unexpected ingredients, or hygienic issues. In addition, 10% of all products labelled as vegetarian contained detectable levels of meat DNA. Vegetarian products also accounted for 67% of hygienic issues, such as human DNA.

At the risk of overly generalizing these findings, I believe that they probably reflect very widespread issues in the food industry, which we as consumers are virtually helpless to deal with, and can only hope that US FDA regulations begin to mandate PCR-based food certification.

Having said this, I didn’t want to end with a “downer.” Therefore, I’ll conclude with some hopefully fun facts about hot dogs, which—if you’re wondering—can be made from beef, pork, turkey, chicken, or a combination (but must be labeled as such, according to FDA information worth reading later).

Fun Frankfurter (aka Hot Dog) Facts

Just for fun, I calculated that the 7 billion frankfurters to-be-consumed by Americans this summer would stretch 56,818 miles (assuming each one were 6 inches long), which would wrap around Earth’s equator 2.3 times if placed end-to-end!

Some other fancinating frankfurter feats:

  • Taken from wikipedia.org

    The world’s longest hot dog was 197 feet and was prepared by Shizuoka Meat Producers and the All-Japan Bread Association for the latter’s 50th anniversary celebration in 2006 at the Akasaka Prince Hotel:

  • The world’s most expensive hot dog is the “California Capitol City Dawg”, served at Capitol Dawg in Sacramento, California and cost $145.49. Proceeds from the sale of each 18-inch long, 3-pound super “Dawg” are donated to the Shriners Hospitals for Children.
  • The annual Nathan’s Hot Dog Eating Contest is held on Independence Day at Nathan’s Famous original, and best-known restaurant in Coney Island, a neighborhood of Brooklyn, New York City. The current champion is Joey “Jaws” Chestnut, who ate a stomach-busting 70 (!) hot dogs and buns in only 10 minutes (!) at the 2016 championship.

Taken from elitedaily.com

As usual, your comments here are welcomed.

Evolving Polymerases to Do the Impossible

  • Polymerases Aren’t What They Used to Be! 
  • Scripps Team Evolves Polymerases That Read and Write With 2’-O-Methyl Ribonucleotides
  • Key Reagents for Romesberg’s “Molecular Moonshots” Are Supplied by TriLink BioTechnologies

Long-time devotees of these posts will likely remember a blog several years ago about Prof. Floyd Romesberg at the Department of Chemistry, The Scripps Research Institute who achieved a seemingly impossible feat. Namely, designing a new pair of complementary bases such that DNA replicating in E. coli would be comprised of six bases, thereby creating a six-base genetic code that is expanded from Nature’s four-base code.

Floyd E. Romesberg. Taken from utsandiego.com

More recently, Romesberg has cleverly outfoxed Nature once again, this time by evolving nucleic acid polymerases into mutant polymerases that can do what heretofore seemed impossible. He and his research team’s publication (Chen et al.) is a tour de force of experimental methodology that is not easily read, and is even harder to simply summarize in a short space like this blog. Consequently, I’ll first tell you what was accomplished, then give a short synopsis of principal new methodology, and close by commenting on the significance of this fascinating work.

Doing the Impossible

Romesberg’s lab successfully achieved what I think of as “multiple molecular moonshots,” wherein a Taq polymerase (which normally reads and writes DNA during PCR), was evolved by novel selection (SELEX) methods into mutant polymerases that are able to transcribe DNA into 2’-O-methyl (2’-OMe) RNA, and reverse transcribe 2’-OMe RNA into DNA for PCR/sequencing.

As depicted below, this was exemplified using a 60-mer DNA template and 18-mer 2’-OMe RNA primer to produce a fully-modified 48-mer 2’-OMe RNA by means of an evolved mPol and all four A, G, C and U 2’-OMe NTPs, which I’m proud to say were bought from TriLink BioTechnologies! This type of molecular evolution of a polymerase has no precedent.

DNA template   5’ ————————————- 3’

RNA primer                                 ←←← 3’ xxxxxx 5’

mPol ↓ 2’-OMe NTPs

Determining the fidelity of this seemingly impossible molecular transformation was addressed by achieving a feat of comparable impossibility! As depicted below, the aforementioned 48-mer 2’-OMe RNA product was hybridized to a DNA primer for reverse transcription into a 48-mer complementary DNA (cDNA) strand, using an evolved mPol, together with all four A, G, C and T unmodified dNTPS, which were also purchased from TriLink. This unprecedented conversion of 2’-OMe RNA into cDNA was followed by conventional PCR/sequencing, the results of which demonstrated relatively high fidelity.

2’-OMe template   5’ xxxxxxxxxxxxxxxxxxxxxxxxxxx 3’

DNA primer                                           ←←← 3’ —— 5’

mPol ↓ dNTPs

cDNA                        3’ ————————————– 5’

How They Did It

In the selection cycle shown below, (1) phage-display libraries were used to expose individual polymerases (Pol) on E. coli. cells in proximity to chemically attached primer/template complexes of interest, which are mixed with natural or modified triphosphates including biotin (green; B)-labelled UTP to extend the primer. (2) Phage that display active mutant polymerases (mPols) are isolated with streptavidin (SA) beads. After washing to remove nonspecific binders, phage cleaved from the beads are used to re-infect E. coli. (3) Heat-treated lysates of E. coli that express the recovered mPols are next subjected to plate-based screening using 96-well plates coated with primer/template complex and extension buffer that contained natural or modified triphosphates and B-UTP, incorporation of which is chromogenically detected. (4) Mutants that give rise to the most activity are selected for individual gel-based analysis, from which (5) promising candidates are selected for further diversification (e.g., by gene shuffling, as depicted) and then subjected to additional rounds of evolution.

Taken from Chen et al. Nature Chemistry (2017)

What is the Significance

In a previous blog, I’ve commented on increasing interest in the utility of aptamers, which are oligonucleotides that can specifically bind small molecules or motifs in proteins, and thus be used to build electronic sensors or studied as potential therapeutic agents rivaling antibodies. Therapeutic aptamers, like antisense oligonucleotides, require incorporation of chemical modifications to impart stability toward nucleases in blood or cellular targets.

Burmeister et al. have previously reported methods for mPol transcription of a DNA template into a fully modified, nuclease-resistant 23-mer 2’-OMe RNA aptamer—also using TriLink’s 2’-OMe NTPs! However, they encountered considerable experimental difficulties in generating this therapeutically promising 23-mer against vascular endothelial growth factor. These technical issues have now been surmounted by the mPol-evolution approaches in the present work by Romesberg’s team, which enabled improved access to longer 2’-OMe RNA aptamers with reasonable efficiency and fidelity.

Moreover, the present study is the first to evolve an mPol for reverse transcription of fully modified 2’-OMe RNA into DNA, which can then be amplified by PCR and/or sequenced, thereby opening the door for a variety of new analytical methods. Most importantly, the molecular mechanism by which these remarkable mPol activities was evolved, namely, the stabilization of an interaction between the “thumb and fingers domains,” may be general and thus useful for the optimization of other Pols. In that case, we can look forward to further advances in evolving other Pols to do the impossible—hopefully using modified nucleotide triphosphates from TriLink!

As usual, your comments are welcome.

Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from gene-quantification.de

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.

Lab-in-a-Box 

In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab

Lab-on-a-Robot

Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.

Lab-on-a-Drone

A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from tamu.edu

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Frightening Fungus Among Us

  • Clinical Alert for Candida auris (C. auris) Issued by CDC
  • US Concerned About C. auris Misidentification and Drug Resistance
  • Sequencing C. auris DNA in Clinical Samples is Preferred for Identification
Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

When I was a kid and didn’t know better, there was a supposedly funny rhyme that “there’s fungus among us.” While this saying is thankfully passé nowadays, the growing number of infections by a formerly obscure but deadly fungus is frightening. This so-called “superbug” is an antibiotic-resistant fungus called Candida auris (C. auris) that’s worth knowing about, and is the fungal focus of this blog.

First, Some Fungus Facts

Fungi are so distinct from plants and animals that they were allotted a biological ‘kingdom’ of their own in classification of life on earth, although that was only relatively recently, i.e. 1969. There are 99,000 know fungi, which exist in a wide diversity of sizes, shapes and complexity that extends from relatively simple unicellular microorganisms, such as yeasts and molds, to much more complex multicellular fungi, such as mushrooms and truffles.

It was previously thought that genomes of all fungi are derived from the genome of the model fungus Saccharomyces cerevisae, which has been used in winemaking, baking and brewing since ancient times. However, genome sequencing of more than 170 fungal species has revealed that, while the genome size of S. cerevisae is only ~12 Mb, seven species of fungus have genome sizes larger than 100 Mb. This is attributed to various evolutionary pressure-factors generating transposable elements, short sequence repeats, microsatellites, and genome duplication, and noncoding DNA.

Fungal cell walls are made up of intertwined fibers mostly comprised of long chains of chitosan, the same tough compound found in the exoskeletons of animals such as spiders, beetles and lobsters. The chitin in fungal cells is entangled with glucans and other wall components, such as proteins, forming a mass that protects the cell membrane behind it—and posing a formidable barrier against antifungal drugs.

Taken from Wikipedia.org

Taken from Wikipedia.org

In researching whether there are any nucleic acid drugs against fungi, I found one early patent by Isis (now Ionis) Pharmaceuticals for use of antisense phosphorothioate-modified oligonucleotides for the treatment of Candida infections, but virtually no other reports. I suspect that will change in the future as pathogenic fungi and other disease-causing microbes become more resistant to conventional drugs.

Fungal infections of the skin are very common and include athlete’s foot, jock itch, ringworm, and yeast infections. While these can usually be readily treated, infections caused by pathogenic fungi have reportedly risen drastically over the past few decades. Moreover, with the increase in the number of immunocompromised (burn, organ transplant, chemotherapy, HIV) patients, fungal infections have led to alarming mortality rates due to ever increasing phenomenon of multidrug resistance.

Segue to a Serious Situation

Emergence of drug-resistant fungi is, in part, the segue to the serious story of the present blog. The other part being incorrect identification of a certain fungus as being a common candida yeast, which is not only scary but seemingly inexcusable in today’s era of highly accurate PCR-based assays to accurately identify microorganisms. Here’s the situation in a nutshell.

  1. auris infection, which is associated with high mortality and is often resistant to multiple antifungal drugs, was first described in 2009 in Japan but has since been reported in countries throughout the world. Unlike many Candida infections, C auris is a hospital-acquired infection that is contracted from the environment or staff of a healthcare facility, and it can spread very quickly.

To determine whether C. auris is present in the United States and to prepare for the possibility of transmission, the Centers for Disease Control (CDC) and Prevention issued a clinical alert in June 2016 requesting that C. auris cases be reported.

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

This official alarm bell, if you will, was triggered by the following facts:

  • Many isolates are resistant to all three major classes of antifungal medications, a feature not found in other clinically relevant Candida
  • auris identification requires specialized methods such as a MALDI-TOF mass spectrometry or sequencing the 28s ribosomal DNA, as pictured below.
  • Using common methods, auris is often misidentified as other yeasts, which could lead to inappropriate treatments.

The CDC subsequently found that seven cases were identified in Illinois, Maryland, New York and New Jersey. Five of seven isolates were either misidentified initially as C. haemulonii or not identified beyond being Candida. Five of seven isolates were resistant to fluconazole; one of these isolates was resistant to amphotericin B, and another isolate was resistant to echinocandins. While no isolate was resistant to all three classes of antifungal medications, emergence of a new strain of C. auris that is would pose a serious public health issue.

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Based on currently available information, the CDC concluded that these cases of C. auris were acquired in the U.S., and several findings suggest that transmission occurred:

  • First, whole-genome sequencing results demonstrate that isolates from patients admitted to the same hospital in New Jersey were nearly identical, as were isolates from patients admitted to the same Illinois hospital.
  • Second, patients were colonized with auris on their skin and other body sites weeks to months after their initial infection, which could present opportunities for contamination of the health care environment.
  • Third, auris was isolated from samples taken from multiple surfaces in one patient’s health care environment, which further suggests that spread within health care settings is possible.

A related Fox News story adds that C. auris was found on a patient’s mattress, bedside table, bed rail, chair, and windowsill. Yikes!

While the above situation in the U.S. might not seem particularly worrisome to you, the potential for emergence of more infectious C. auris strains with higher lethality should be of concern. That has already reportedly occurred in several Asian countries and South Africa. Obviously, deployment of the best available methods for pathogen identification can, in principle, lessen the likelihood of the emergence and/or spread of C. auris in the U.S. and other countries.

Case for Point-of-Care C. auris Nanopore Sequencing?

Taken from extremtech.com 

Taken from extremtech.com

Regular readers of my previous blogs know that I’m an enthusiastic fan of the Oxford Nanopore Technologies minION sequencer, which is proving to be quite useful for characterizing pathogens in very remote regions on Earth—and even on the International Space Station to diagnose astronaut infections! Notwithstanding various current limitations for minION sequencing of microbes, it seems to me that it would be relatively straightforward to generate minION data for many available samples of pathogenic fungi and genetically related microbes to assess the feasibility using minION for faster, cheaper, better unambiguous identification of C. auris minION in centralized or Point-of-Care applications.

Taken from rnaseq.com

Taken from rnaseq.com

If you think this suggestion is farfetched, think again, after checking out these 2016 publications using minION:

The 51.4-Mb genome sequence of Calonectria pseudonaviculata for fungal plant pathogen diagnosis was obtain using minION.

The first report of the ~54 Mb eukaryotic genome sequence of Rhizoctonia solani, an important pathogenic fungal species of maize, was derived using minION.

Sequence data is generated in ~3.5 hours, and bacteria, viruses and fungi present in the sample of marijuana are classified to subspecies and strain level in a quantitative manner, without prior knowledge of the sample composition.

CDC on C. auris Status and FAQs

In the interest of concluding this blog with the most up-to-date and authoritative information, I consulted the CDC website and found statements and replies to FAQs that are well worth reading at this link.

As a scientist, my overriding question concerns the lack of adoption of improved microbiological methods by hospitals and clinics. The above noted misidentifications of C. auris infections resulting from use of flawed lab analyses seems unacceptable. Although I don’t know all the facts or statistics to generalize, I suspect that there are other incorrect lab analyses due to use of outdated methods. On the other hand, I’m hopeful that, with the FDA’s widely touted Strategic Plan for Moving Regulatory Science into the 21st Century, the section entitled Ensure FDA Readiness to Evaluate Innovative Emerging Technologies—think nanopore sequencing—becomes actionable, sooner rather than later.

Changing established—dare I say entrenched—clinical lab tests is not simple or easy, but if it doesn’t begin it won’t happen, about which I’m quite certain. I can only wonder why development of infectious disease analytical methods and treatments seem to require a crisis. Sadly, I think it boils down to the complexities and socio-political dynamics of who pays.

Frankly, it’s my personal opinion that maybe it’s time Thomas Jefferson’s philosophy about hammering guns into plows is directed to health care.

Postscript

After writing this blog, I learned that T2 Biosystems has received FDA approval to market in the U.S. the first direct blood test for detection of five yeast pathogens that cause bloodstream infections: Candida albicans and/or Candida tropicalis, Candida parapsilosis, Candida glabrata and/or Candida krusei.

Yeast bloodstream infections are a type of fungal infection that can lead to severe complications and even death if not treated rapidly. Traditional methods of detecting yeast pathogens in the bloodstream can require up to six days, and even more time to identify the specific type of yeast present. The T2Candida Panel and T2Dx Instrument (T2Candida) can identify these five common yeast pathogens from a single blood specimen within 3-5 hours.

T2Candida incorporates technologies that break the yeast cells apart, releasing the DNA for PCR amplification for detection by greatly simplified, miniaturized nuclear magnetic resonance (NMR) technology, as can be seen in this video.

In my opinion, this fascinating new technology is another example of what could be rapidly deployed toward detecting C. auris.

Update on Zika Virus Detection by RT-PCR

  • Various RT-PCR Assays for Zika Virus Have now Received Emergency Use Authorization (EUA) from the FDA
  • Quest Diagnostics’ Assay Approved by FDA for General Use as a Zika Test
  • Troubled Theranos Touts New “miniLab” for Zika EUA
  • Vaccine Development Progressing Albeit Relatively Slowly
Aedes aegypti mosquito. Taken from wcvb.com

Aedes aegypti mosquito. Taken from wcvb.com

In January 2016, I posted a blog about the then emerging public awareness of Zika virus (ZIKV), which is spread by the bite of infected mosquitos—primarily the Aedes aegypti mosquito. Sadly, ZIKV can be passed from a ZIKV-infected pregnant woman to her fetus leading to development of a brain defect (microcephaly) and/or other malformities. Moreover, ZIKV is now associated with sexual transmission and blood transfusion. This is scary news.

This update was prompted by the additional fact that ZIKV is on the rise and there is still no vaccine, according to the Centers for Disease Control and Prevention (CDC)—my “go to” source for loads of authoritative information about this infectious disease. Weekly updated ZIKV-infection “case counts” in US States and US Territories were given as ~1,200 and ~6,500, respectively on Aug 10th, and increasing to ~3,400 and ~20,000 on Sep 21st —only 6 weeks later!

As I pointed out in my previous blog on ZIKV, absent an anti-ZIKV vaccine, there is considerable interest in mosquito abatement as well as early detection, notably by reverse-transcription PCR (RT-PCR) of this RNA Flavivirus. Now that Zika infection by mosquitos in Florida has been found, leading to several CDC warnings, I thought it would be both apropos and technically interesting to provide the following update on ZIKV structure and RT-PCR.

ZIKV Genome and Structure

As of last month, my PubMed search of “Zika [Title/Abstract] AND RT-PCR [Anywhere]” gave 55 publications. Incidentally, there are now a number of Zika genome sequencing publications, such as this lead reference. It’s worth noting that ZIKV genome sequencing enables monitoring the potential evolution of new genomic variants that might foil existing RT-PCR assays and guide the selection of new primers for RT-PCR.

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

It’s also worth pointing out that Zika’s overall molecular capsid structure is enveloped, spherical, and has a diameter of ~40 nm in diameter, as depicted below in schematic form, and pictured in the accompanying electron microscopic image. The surface proteins are arranged in an icosahedral-like symmetry.

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

ZIKV RT-PCR

In the interest of giving explicit credit to various investigative groups who have developed RT-PCR assays for ZIKV, here are links and short snippets I’ve selected from publications, in chronological order, found in the aforementioned PubMed search. Interested readers are encouraged to check out the original publication for details.

The first report of an RT-PCR assay for ZIKV appears to be by Faye et al. in 2008 at the Institut Pasteur de Dakar in Senegal, who targeted the envelope protein coding region and tested ZIKV isolates previously collected over a 40-year period from various African countries and hosts. The assay’s detection limit and repeatability were respectively 7.7pfu/reaction and 100% in serum; none of 19 other Flaviviruses tested were detected. Faye et al. in 2013 extended this work to include quantitative RT-PCR detection of ZIKV and evaluation with samples from field-caught mosquitoes.

Kinetics of ZIKV detection in urine compared to serum from 6 patients was described by Gourinat et al. in 2015 at the Institut Pasteur, Noumea, New Caledonia using RT-PCR primers and probes previously reported by others. Urine samples were positive for ZIKV more than 10 days after onset of disease, which was a notably longer period than 2-3 days for serum samples. These researchers concluded that urine samples are useful for diagnosis of ZIKV infections, and are preferred to serum wherein virus titer diminishes more rapidly.

Musso et al. in 2015 working in Tahiti, French Polynesia with 1,067 samples collected from 855 patients presenting symptoms of Zika fever found that analysis of saliva samples increased the rate of detection of ZIKV at the acute phase of the disease compared to serum samples. They noted that saliva was of particular interest when blood was difficult to collect, especially for children and neonates.

Most recently, Xu et al. in 2016 in China reported the development of a SYBR Green (intercalator dye)-based qRT-PCR assay for detection of ZIKV. Although their results indicate that the assay is specific, it’s important to note that SYBR-type detection can be subject to nonspecific artifacts, for which TriLink’s proprietary CleanAmp™ Primers can be investigated to potentially ameliorate such problems, as discussed in this downloadable pdf publication by TriLink researchers.

ZIKV In Vitro Diagnostic Assays

As detailed elsewhere, the Secretary of Health and Human Services (HHS) earlier this year determined that “there is a significant potential for a public health emergency that has a significant potential to affect national security or the health and security of United States citizens living abroad and that involves Zika virus.” The Secretary of HHS further declared that “circumstances exist justifying the authorization of the emergency use of in vitro diagnostics for detection of Zika virus…”.

The following RNA-based assays and suppliers are currently listed by the FDA for this Emergency Use Authorization (EUA):

  • xMAP® MultiFLEX™ Zika RNA Assay (Luminex Corporation)
  • VERSANT® Zika RNA 1.0 Assay (kPCR) Kit (Siemens Healthcare Diagnostics Inc.)
  • Zika Virus Real-time RT-PCR Test (Viracor-IBT Laboratories, Inc.)
  • Aptima® Zika Virus Assay (Hologic, Inc.)
  • RealStar® Zika Virus RT-PCR Kit U.S. (Altona Diagnostics)
  • Zika Virus RNA Qualitative Real-Time RT-PCR (Focus Diagnostics)
  • Trioplex Real-time RT-PCR Assay (CDC)

Among these, two are particularly notable—in my opinion. Trioplex Real-time RT-PCR Assay is a multiplexed laboratory test developed by the CDC to simultaneously detect ZIKA, dengue virus, and chikungunya virus RNA, each of which can be transmitted primarily by Aedes aegypti mosquitos to cause infections with similar symptoms. Consequently, it is useful to have a single test that can detect each of these viruses in the same sample. Full details for the Trioplex assay are provided in a 40-page downloadable pdf from the CDC at this link.

In brief, this CDC-developed test uses virus-specific primer pairs and fluorogenic hydrolysis probes each differentially dual-labeled with fluorescent reporter and quencher dyes for in vitro detection of complementary DNA (cDNA). The detection follows reverse-transcription of RNA isolated from clinical specimens including serum, cerebral spinal fluid, urine, and amniotic fluid. It’s worth pointing out that I subsequently found a publication in 2016 by several collaborating academic groups regarding an analogous triplex RT-PCR assay for the same three viruses.

The other notable assay is Zika Virus RNA Qualitative Real-Time RT-PCR developed by Focus Diagnostics, which in April 2016 was the first of the aforementioned ZIKV tests to receive authorization by the FDA for use by qualified labs to detect ZIKV RNA in blood samples of those meeting CDC clinical criteria or of people who may have lived in or traveled to an affected location or had other exposure to the virus. Quest Diagnostics, the parent company of Focus Diagnostics, announced it would make the test broadly available to physicians, including those in Puerto Rico in May 2016.

ZIKV EUA Sought by Theranos for Its new “miniLab”

Theranos, which has been in the news regarding troubles over its stealthy proprietary system for finger-stick blood tests, appears to be pivoting its strategic plans. It announced at the August 2016 American Association of Clinical Chemistry Meeting its R&D for a new, fully automated miniLab system, including analytical and method comparison results of its ZIKV nucleic acid-amplification-based assay.

According to its press release, the company collected finger-stick samples from subjects, including people in the ZIKV-infested Dominican Republic, and shipped those to Palo Alto, California to run on the miniLab. Although I was unable to obtain these particular results, I did find the following figure at the TechCrunch website showing functional components of the miniLab along with an article by Sarah Buhr that’s worth a quick read, in my opinion.

Taken from techcruch.com

Taken from techcruch.com

According to the aforementioned Theranos press release, the company has submitted assay validation data for this Zika assay to the FDA for an EUA. The company also states that it is unaware of any currently available capillary (i.e. finger-stick) test for ZIKV.

I’ll stay tuned for future general information about the miniLab, as well as information specifically related to ZIKV. If I hear of anything, I’ll add as a comment here or in a new post with technical details about its nucleic acid assays.

ZIKV Vaccine Status

I hope this blog has convinced you that RT-PCR of ZIKV has provided improved molecular diagnostics. I’m guessing, that like me, you find it unfortunate that there isn’t a proven anti-ZIKA vaccine as of yet. This is especially frustrating given the fact that Zika disease has been known for more than 50 years, and that it is evidently on the rise globally, including in the USA according to regularly updated CDC statistics.

In February 2016, the Obama administration requested $1.9 billion in funding for the NIH to develop a ZIKV vaccine. The US Congress continues to be deadlocked by partisan politics despite the fact that Florida state and local officials are scrambling to contain the ZIKV outbreak in Miami Beach. This outbreak poses a serious threat to the health of residents, as well to visitors who drive the region’s $24 billion-a-year tourism industry.

Nevertheless, some progress has been reported for US studies in monkeys, and US-based Inovio says it’s received FDA approval to begin studies in humans. Outside the US, Sanofi Pasteur in France is said to be poised for initiating its trials in humans, and French biotech company Valneva is reported to have succeeded in generating a ‘highly purified inactivated vaccine candidate’ using the same technical approach it used for its encephalitis vaccine that is marketed in the United States and Europe.

Lagging—dare I say “glacially slow”—action against ZIKV by the World Health Organization is quite disappointing to me, and is reminiscent of what I’ve commented on in an earlier blog concerning this bureaucracy’s ineffective response to the Ebola virus. If I had my druthers, TriLink’s previously announced engagement by Battelle in development of Ebola mRNA vaccine would somehow materialize for a Zika mRNA vaccine. In this regard, GlaxoSmithKline is reported to be preparing research studies alongside the NIH’s Vaccine Research Center to test a self-amplifying mRNA vaccine technology for Zika. Interested readers can check out this link to a fascinating PNAS publication by Geall et al. on biosynthetic self-amplifying mRNA vaccines delivered in lipid nanoparticles.

As always, your comments are welcomed.

Postscript

Taken from Fleming et al. (2016) ACS Infectious Diseases

Taken from Fleming et al. (2016) ACS Infectious Diseases

One of my previous posts featured recent elucidation of biologically functional G-quadraplexes in living cells. Consequently, it’s apropos to mention here that Fleming et al. at the University of Utah have just now published the first analysis of potential G-quadruplex sequences (PQS) in the RNA genome of ZIKV. As depicted in this artistic cartoon, several PQS were found, with the most stable located near the end of the 3’ untranslated region (3′-UTR). Importantly, these investigators propose a rationale for screening G-quadruplex-binding compounds as a completely new class of anti-ZIKV drug candidates. In my opinion, this is a great example of how basic biochemical research can lead to new strategies for much needed antiviral drugs.

RNA World Revisited

  • Scripps Researchers ‘Evolve’ an RNA-Amplifying RNA Polymerase 
  • It’s Used for First Ever All-RNA Amplification Called “riboPCR”
  • TriLink Reagent Plays a Role in this Remarkably Selective in Vitro Evolution Method 
Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from scripps.edu

Prof. Gerald Joyce & Dr. David Horning. Photo by Madeline McCurry-Schmidt. Taken from scripps.edu

Those of you who regularly read my blog will recall an earlier posting on “the RNA World,” which was envisioned by Prof. Walter Gilbert in the 1980s as a prebiotic place billions of years ago when life began without DNA. That post recommended reading more about this intriguing hypothesis by consulting a lengthy review by Prof. Gerald Joyce. Now, Prof. Joyce and postdoc David Horning have advanced the hypothesis one step further by reporting the first ever amplification of RNA by an in vitro-selected RNA polymerase, thus providing significant supportive evidence for the RNA World. Following are their key findings, which were enabled in part by a TriLink reagent—read on to find out which one and how!

In Vitro Evolution of an RNA Polymerase

Horning & Joyce designed an in vitro selection method to chemically “evolve” an RNA polymerase capable of copying a relatively long RNA template with relatively high fidelity. The double emphasis on “relatively” takes into account that the RNA World would have many millions of years to evolve functionally better RNA polymerases capable of copying increasingly longer RNA templates with increasingly higher fidelity.

As depicted below, they started with a synthetic, highly structured ribozyme (black) wherein random mutations were introduced throughout the molecule at a frequency of 10% per nucleotide position to generate a population of 1014 (100,000,000,000,000) distinct variants to initiate the in vitro evolution process. Step 1 involved 5’-5’ click-mediated 1,2,3-trazole (Ø) attachment of an 11-nt RNA primer (magenta) partially complementary to a synthetic 41-nt RNA template (brown) encoding an aptamer that binds guanosine triphosphate (GTP). In Steps 2 and 3, the primer hybridizes to template and is extended by polymerization of A, G, C and U triphosphates (cyan).

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

GTP aptamer showing red and cyan sequences corresponding to above cartoon. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Step 4 involves binding of aptameric structures to immobilized GTP (green), then photocleavage of the 1,2,3-triazole linkage in Step 5, followed by reverse transcription to cDNA and conventional PCR in Step 6 for transcription into ribozymes in Step 7. Twenty-four rounds of this evolution by selection were carried out, progressively increasing the stringency by increasing the length of RNA to be synthesized by decreasing the time allowed for polymerization. By the 24th round, the population could readily complete the GTP aptamer shown below. Subsequent cloning, sequencing and screening were then used to characterize the most active polymerase, which was designated “24-3.”

The TriLink “Connection”

2'-Azido-dUTP (aka 2'-azido-UTP)

2′-Azido-dUTP (aka 2′-azido-UTP)

The aforementioned in vitro evolution process actually involves tons of experimental details that interested readers will need to consult in the published paper, which is accompanied by an extensive Supporting Information section. In the latter, a subsection titled Primer Extension Reaction describes 3’ biotinylation of the template RNA strand (brown in above scheme) using TriLink “2’-azido-UTP” (more properly named 2’-azido-dUTP) and yeast poly(A) polymerase, followed by click connection of the RNA template’s 3’-terminal 2’-azido moiety to biotin-alkyne. This very clever functionalization of the RNA template strand allowed for subsequent capture of the double-stranded primer extension reaction products on streptavidin-coated beads, followed by elution of the desired nonbiotinylated strand for GTP aptamer selection (Step 4 above).

Properties of RNA Polymerase 24-3

Needless to say—but I will—enzymologists and RNA aficionados will undoubtedly be interested in musing over the kinetic and fidelity properties of RNA polymerase 24-3.

The rate of 24-3 polymerase catalyzed addition to a template-bound primer was measured using an 11-nt template that is cited extensively in the literature to evaluate various ribozymes. It was found that the average rate of primer extension by 24-3 is 1.2 nt/min, which is ∼100-fold faster than that of the starting ribozyme polymerase randomly mutagenized for in vitro selection.

The NTP incorporation fidelities of the starting and 24-3 ribozyme polymerases on this 11-nt test template, at comparable yields of product, are 96.6% and 92.0%, respectively. Horning & Joyce noted that the higher error rate of 24-3 is due primarily to an increased tendency for G•U wobble pairing.

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Phenylalanyl tRNA. Taken from Horning & Joyce, Proc. Natl. Acad. Sci., 2016

Other longer RNA templates having various base compositions or intramolecular structures were also studied, with the stated “final test of polymerase generality” being use of 24-3 to synthesize yeast phenylalanyl tRNA from a 15-nt primer (in red right). The authors humorously describe the results as follows:

“Despite the stable and complex structure of the template, full-length tRNA was obtained in 0.07% yield after 72 h. This RNA product is close to the limit of what can be achieved with the polymerase, but is likely the first time a tRNA molecule has been synthesized by a ribozyme since the end of the RNA world, nearly four billion years ago.”

Exponential Amplification of RNA

PCR is the most widely used method for amplifying nucleic acids, and involves repeated cycles of heat denaturation and primer extension. The 24-3 RNA polymerase was used to carry out PCR-like amplification, but in an all-RNA system (named riboPCR by Horning & Joyce) using A, G, C, and U triphosphates and a 24-nt RNA template composed of two 10-nt primer-binding sites flanking the sequence AGAG. Somewhat special conditions were employed:

  • The concentration of Mg2+ was reduced to minimize spontaneous RNA cleavage
  • PEG8000 was used as a “molecular crowding” agent to improve ribozyme activity at the reduced Mg2+ concentration
  • Tetrapropylammonium chloride was added to lower the melting temperature of the duplex RNA

Under these conditions, 1 nM of the 24-nt RNA template was driven through >40 repeated thermal cycles, resulting in 98 nM newly synthesized template and 106 nM of its complement, corresponding to 100-fold amplification. Sequencing of the amplified products revealed that the central AGAG sequence was largely preserved, albeit with a propensity to mutate the third position from A to G, reflecting the low barrier to wobble pairing.

Amplification of a 20-nt template (without the central insert) was monitored in real time, using FRET from fluorescently labeled primers, and input template concentrations ranging from 10 nM to 1 pM. The resulting amplification profiles shown in the paper are typical for real-time PCR, shifted by a constant number of cycles per log-change in starting template concentration. A plot of cycle-to-threshold vs. logarithm of template concentration, also shown in the paper, was linear across the entire range of dilutions indicating exponential amplification of the template RNA with a per-cycle amplification efficiency of 1.3-fold.

Implications for the Ancient RNA World

It would be an injustice to Horning & Joyce if I would try to paraphrase their concluding discussion of this investigation, so here is what they say:

The vestiges of the late RNA world appear to be shared by all extant life on Earth, most notably in the catalytic center of the ribosome, but most features of RNA-based life likely were lost in the Archaean era. Whatever forms of RNA life existed, they must have had the ability to replicate genetic information and express it as functional molecules. The 24-3 polymerase is the first known ribozyme that is able to amplify RNA and to synthesize complex functional RNAs. To achieve fully autonomous RNA replication, these two activities must be combined and further improved to provide a polymerase ribozyme that can replicate itself and other ribozymes of similar complexity. Such a system could, under appropriate conditions, be capable of self-sustained Darwinian evolution and would constitute a synthetic form of RNA life.

Applications for Today’s World of Biotechnology

The aforementioned report by Horning & Joyce has received wide acclaim in the scientific press and world-wide public media as supporting the existence of a prebiotic RNA World, billions of years ago, from which life on Earth evolved.

While the academic part of my brain, if you will, fully appreciates the significance of these new insights on “living” RNA eons ago, the technical applications part of my brain is more piqued by possible practical uses of all-RNA copying or all-RNA riboPCR.

I, for one, plan to muse over possible applications of such all-RNA systems in today’s world of biotechnology, and hope that you do too, and are willing to share any ideas as comments here.