Clinical Efficacy Found: Two Oligonucleotide Drugs for Transthyretin (TTR)-Related Amyloidosis (ATTR)

  • ATTR Is a Class of Life-Threatening and Progressive Genetic Diseases    
  • ATTR Results from Misfolding of TTR, a Blood Protein Carrier of Retinol (Vitamin A), and Thyroxine (T4)
  • TTR mRNA Knockdown by an Antisense Oligonucleotide (ASO) or a Short-Interfering RNA (siRNA) Leads to Clinical Efficacy for ATTR

A recent blog here lauded the beginning of American and European clinical studies of gene-editing by CRISPR, which is enabled by sequence-specific interaction of synthetic guide RNAs with genomic DNA targets. At the end of that post, I mentioned that future clinical successes with CRISPR may encounter the challenge of unforeseen drug development issues, as has been the case for antisense oligonucleotide (ASO) and short-interfering RNA (siRNA) approaches. It took a couple of decades for FDA-approved ASO drugs to progress from “bench to bedside,” and the first siRNA drug has yet to be approved.

After opining the above just a few weeks ago, I am pleased to say that another ASO drug and the first siRNA drug have been reported to both be on the brink of FDA approval, based on back-to-back publications of the results of Phase 3 clinical studies in the highly respected Journal of New England Medicine. Interestingly, both of these mechanistically different oligonucleotide drugs are aimed at the same clinical indication, namely, transthyretin (TTR)-related amyloidosis (ATTR). This blog will serve as an introduction to ATTR, as well as a brief summary of both of these very promising clinical studies.

Introduction to ATTR

As detailed elsewhere, ATTR represents a class of life-threatening and progressing diseases that is associated with the misfolding of TTR, a major blood protein that is a carrier of retinol (vitamin A) and thyroxine (thyroid hormone T4). TTR is primarily (>95%) produced in the liver as a tetramer, as depicted below. Amyloid aggregation is believed to be a result of decreased tetramer stability, resulting in dissociation of TTR into monomers. These monomers are prone to unfolding, and can self-assemble into oligomers and amyloid fibrils found in peripheral neurons, the gastrointestinal tract, and heart.

Taken from

The three major forms of ATTR disease are described in the NIH Genetic Home Reference (GHR), which also provides information about their underlying genetics and diagnosis by, for example, DNA sequencing. In hereditary ATTR with polyneuropathy [aka familial amyloid polyneuropathy (FAP)], the peripheral nerves are primarily affected, while in cardiomyopathy-related ATTR [aka familial amyloid cardiomyopathy (FAC)], neuropathy is usually less prominent.

According to the GHR, ATTR among Americans of European descent is estimated to affect one in 100,000 people, while FAC is far more frequent among people with African ancestry. For example, it is estimated that this form affects between three to four in 100 African Americans. Although the reasons for this disproportionately higher incidence are unknown, suggestions on how to raise awareness on this issue are discussed in a review I found while researching FAC among African Americans.

For the most part, patients develop a severe disease and die within 5 to 15 years after onset. While these forms of ATTR can be ascribed to a dominant expression of the TTR gene variants, (their number is ~100, as depicted here in an artistic rendering), only wild-type TTR is expressed in senile systemic amyloidosis (SSA), a type of amyloidosis frequently found in elderly people.

Taken from

The ATTR pathway above depicts the possibility of using oligonucleotides that interfere with mRNA function to block the synthesis of TTR. While blocking synthesis of a specific mutant of TTR is possible, biochemical and clinical evidence suggests that the wild-type TTR can also significantly contribute to the disease. In 2016, results obtained from an in vitro model system were jointly published by Ionis Pharmaceuticals and Alnylam Pharmaceuticals. The study supports the notion that ASOs or siRNAs can block TTR synthesis, and can therefore represent a possible therapy for ATTR. These findings paved the way for each company to independently carry out individual clinical development programs for oligonucleotide drug candidates, as outlined below.

ASO Drug Inotersen

Ionis’ inotersen (formerly IONIS-TTRRx/ISIS 420915), which is a 2′-O-methoxyethyl (MOE)– and phosphorothioate (PS)-modified RNA (orange)-DNA (black) “gapmer” ASO shown here, is an inhibitor of the hepatic production of TTR. Readers interested in ASO chemistry and mechanistic details for gapmers can consult an excellent review by Shen and Corey.

Taken from Shen and Corey Nucleic Acids Research (2018)

Taken from Buxbaum Journal of New England Medicine (2018)

As shown here, the genome of patients with hereditary amyloidotic polyneuropathy contains one copy of wild-type TTR and one copy with a base substitution (mutation), resulting in a change in the amino acid sequence. In hepatocytes, both copies are equally transcribed and translated. Inotersen binds to wild-type and mutant TTR mRNA transcripts, resulting in their degradation by ribonuclease H.

In healthy volunteers, inotersen showed dose-dependent and sustained reductions of circulating TTR levels. Ionis then conducted a randomized, double-blind, placebo-controlled, Phase 3 trial (NEURO-TTR) to determine the efficacy and safety of inotersen treatment in patients with hereditary transthyretin amyloidosis with polyneuropathy, in the presence or absence of cardiomyopathy. According to Benson et al., eligible patients were randomly assigned (in a 2:1 ratio) to receive 300 mg of inotersen or placebo. Patients 18 to 82 years of age received three subcutaneous injections during the first week to achieve near steady-state drug levels, followed by a once-weekly subcutaneous injection for the next 64 weeks.

A total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. In the inotersen group, reductions in circulating TTR reached steady-state levels by week 13 and were sustained through the end of the intervention period. From week 13 to week 65, decreases in serum TTR from baseline levels in the inotersen group reached a median nadir of 79%.

Primary efficacy assessments favored inotersen. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. The most frequent serious adverse events in the inotersen group were glomerulonephritis [in 3 patients (3%)] and thrombocytopenia [in 3 patients (3%)]. It was concluded that “inotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis” and that “[t]hrombocytopenia and glomerulonephritis were managed with enhanced monitoring.”

siRNA Drug Patisiran

According to the above cited review by Shen and Corey, and as shown here, Alnylam’s patisiran is a double-stranded siRNA comprised of 3’-end DNA (black), RNA (pink), and 2’-O-MOE RNA (blue) moieties. Partisiran is delivered in complex with a lipid nanoparticle (LNP), (also depicted here), and is described in detail elsewhere by Acuitas Therapeutics, an LNP drug-delivery company that partnered with Alnylam.

Taken from Shen and Corey Nucleic Acids Research (2018)

Taken from

The mechanism of action depicted here for the siRNA patisiran involves the formation of an RNA-induced silencing complex (RISC) with both wild-type and mutant TTR mRNA transcripts and subsequent mRNA degradation.

Taken from Buxbaum Journal of New England Medicine (2018)

Adams et al. have now reported results from a Phase 3 clinical trial by Alnylam wherein patients were randomly assigned (in a 2:1 ratio) to receive patisiran (0.3 mg per kilogram of body weight) or placebo intravenously over a period of approximately 80 minutes, once every 3 weeks for 18 months. A total of 225 patients 24 to 83 years of age were randomly assigned to receive patisiran (148 patients) or placebo (77). In the patisiran group, the reduction in serum TTR levels was rapid and sustained over a period of 18 months, with a median reduction of 81% that remained similar across age, sex, or genotype.

Primary efficacy endpoints favored patisiran and approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups. It was concluded that “patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis.”

One Expert’s Opinion

Joel Buxbaum, an MD and Professor Emeritus in the Department of Molecular Medicine at the Scripps Research Institute, accompanied these back-to-back publications about inotersen and patisiran in the Journal of New England Medicine with an Editorial. Based on his publications in this field, Buxbaum is an expert on the molecular basis of acquired and hereditary human disease related to human amyloidosis. After summarizing the results of each clinical trial, Buxbaum posed the following scientifically intriguing questions, which had not occurred to me after reading these two reports.

“Although each of the trials unequivocally shows a therapeutic effect, there remain questions. Would a single patient have a response to each of the therapeutics to the same degree? If so, it is in the patient’s interest to use the least expensive therapy. If not, N-of-1 trials of sequential treatments, although cumbersome, would be required to identify the best treatment for each patient, conditional on the availability of a rapidly responsive, validated surrogate marker of disease.” In regard to the currently trending concept of N-of-1 trials, Buxbaum refers the reader to a commentary in Nature by Nicholas Schork titled Personalized medicine: Time for one-person trials, from which I borrowed this illustration that visually coveys Schork’s point.

Taken from Schork Nature (2015). Illustration by Greg Clarke

Buxbaum continues his expert commentary as follows:

“It is also possible that a combination of interventions would elicit a more pronounced, durable therapeutic effect. Although there remains much work to be done, the trials by Adams et al. and Benson et al. represent a landmark: together, they show that the rate of progression of a peripheral neurologic disease can be slowed, and perhaps ameliorated, through the use of oligonucleotide drugs that are administered systemically.”

In my humble opinion, the promising outcomes of these two trials are indeed great news for people afflicted with ATTR, who can be future recipients of this oligonucleotide therapy. The results are likewise great news for families and friends. The many researchers, who collectively contributed—directly or indirectly—to achieving this milestone, are also undoubtedly pleased by these results.

As usual, your comments are welcomed.

Reported: First Visualization of I-Motif DNA in Human Cells

  • Although Akin to Well-Known G-Quadruplexes, I-Motif DNA In Vivo Has Been Debatable
  • Now, Researchers Have Reported the First Visualization of I-Motifs in Human Cells Using an Antibody
  • These Researchers Raise Questions About Antibody Specificity for Visualizing G-Quadruplex vs. I-Motif Structures

In April 2013, in my second ever blog, DNA G-quadruplexes (GQs) were featured as a then trending hot topic in nucleic acid research, following a report in Nature Chemistry of the first visualization of these DNA structures in human cells. That published work, led by University of Cambridge Prof. Shankar Balasubramanian, has been cited nearly 2,000 times. This puts it in the 99th percentile (ranked 160th) of the 309,600 tracked articles of a similar age in all journals, according to an Altmetric score.

Sir Shankar (©Caroline Hancox). Taken from

The aforementioned 2013 blog includes an interview with Prof. Balasubramanian, who was knighted in January 2017 in part for that pioneering work. Although somewhat akin to GQs, iM DNA has eluded detection in vivo until now. Aussie researchers have visualized iM DNA for the first time, as described in an April 2018 publication in Nature Chemistry. Following my brief synopsis of these new findings, Sir Shankar was kind enough to share some additional perspectives on iMs with us for discussion.

Introduction to GQ and iM DNA Structures

DNA is well-known to adopt alternative non-B-form conformations in vitro, including GQ (aka G4) and iM structures, which are depicted here in a figure taken from the aforementioned Nature Chemistry report in 2018 by the Aussie team of Zeraati et al. Of the two structures, GQ DNA formed within guanine (G)-rich regions of the genome is by far the more studied. In spite of extensive bioinformatic and in vitro characterizations of GQs, the in vivo existence of GQs in human cells remained speculative until the visualization of these structures by Sir Shankar’s team. The team used an antibody fragment that recognizes GQs (shown in red) in a structure-specific manner.

Taken from Zeraati et al. Nature Chemistry (2018)

In marked contrast, insights into the biological role of iM DNA are limited. This structure is formed via a stack of intercalating hemi-protonated C—neutral C base pairs (shown in green), which are stabilized at a slightly acidic pH. As C-rich regions are common within the human genome and can occur opposite G-rich regions (hence the interconversion arrows in the figure), iMs have been characterized in vitro using a range of biophysical techniques. These analyses demonstrated the formation of both intramolecular (as depicted in the figure) and intermolecular structures, with the overall stability of the structure dependent on the number of Cs in the iM core, as well as the length and composition of the loops.

However, the in vivo existence of this four-stranded iM DNA structure in the human genome has been a matter of scientific debate. In particular, observations that in vitro formation of the iM structure is dependent on acidic conditions has led to questions concerning biological relevance. But now, as you’ll read in what follows, such doubt about the biological relevance of iM DNA has been countered by compelling evidence in support of the in vivo existence of iM DNA, and suggestions of regulatory roles for iM DNA in the genome.

An Antibody Fragment Specific for iM Structures

The recent publication by Zeraati et al. should be read in its entirety to fully appreciate the methodology used to isolate an iM-specific antibody fragment for the study of iM structures in vitro and in vivo. Briefly, three rounds of phage-display selections were performed to isolate iM DNA-binders. The binders were then initially characterized using soluble fragment enzyme-linked immunosorbent assay (ELISA). This was followed by evaluation of the bio-layer interferometry (BLI) off-rate ranking, resulting in the identification of a lead candidate, which was termed iMab.

Diagram of BLI activity with immobilized binding partner attached. Taken from

Further ELISA analyses revealed highly specific binding, as indicated by the absence of iMab binding to a wide range of control proteins and nucleic acids, including double-stranded DNA, hairpin DNA, and microRNA. To investigate whether iMab is capable of binding to partially folded iM structures, Zeraati et al. tested truncated constructs with either three or two tandem runs of Cs. The two truncated constructs displayed either considerably reduced binding (three C-runs) or no detectable binding (two C-runs), indicating that recognition by iMab is highly dependent on the folded structural framework of C-containing DNA.

The pH-dependence of iM stability and iMab binding was investigated because iM structures are considered most stable at acidic pH, reflecting the requirement for the formation of hemi-protonated C—neutral C base pairs (see above). Zeraati et al. thus hypothesized that pH and the binding of iMab should be inversely correlated, assuming that the antibody predominantly recognizes folded iM rather than unfolded species. Using BLI and a constant concentration of iMab binding to an iM structure, it was found that the iMab-iM complex antibody formed at pH 6, and could be readily dissociated when switching to pH 9 buffer conditions.

iMab Differentiates iM Structures from GQ Structures

Zeraati et al. reasoned that iMs and GQs could display common epitopes, i.e. a specific piece of DNA to which iMab binds. Moreover, in genomic DNA, iMs are often accompanied by complementary G-rich sequences, which can adopt GQ structures as depicted above. For applications such as visualization by immunostaining, a high level of specificity and the absence of binding to GQs is therefore important. To evaluate iMab cross-reactivity, Zeraati et al. examined binding to six GQs of known structures, which were selected to represent a diverse range of conformations. ELISA results showed that iMab does not display any detectable binding to these molecules, suggesting that it is highly capable of differentiating between GQ and iM structures.

The studied presumptive negative controls included commercially available GQ antibody BG4, which was initially described by Sir Shankar and coworkers in Nature Chemistry as noted above. BG4 showed substantial binding to all analyzed GQ structures, as expected; however, BG4 also displayed detectable cross-reactivity to iMs and competed with iMab for binding to several iMs. Given the potential complications that could result from BG4 cross-reactivity with iMs, independent confirmatory investigations are needed to address the nature and scope of this putative cross-reactivity reported by Zeraati et al.

iM Structures are Formed in the Nuclei of Human Cells

Zeraati et al. used iMab for indirect immunofluorescent staining of three different human cell lines. This staining revealed punctate foci in the nuclei of MCF7, U2OS and HeLa cell lines that were dependent on the presence of iMab, as shown here for MCF7 cells. In addition, DNase I treatment significantly decreased the number of foci in the nuclei, presumably due to nuclease-resistant DNA structures or shielding by proteins. Zeraati et al. speculated that another possibility was the formation of iM RNA structures that are not removed by DNase I digestion. A small decrease in the number of iMab foci after RNase A treatment supported this speculation.

Imaging of iMab foci in the nuclei of MCF7 cells using confocal microscopy. Nuclei were counterstained with DAPI. Yellow dashed lines in the DNase I treated samples indicate the borders of nuclei. Scale bars, 5 μ m. Taken from Zeraati et al. Nature Chemistry (2018)

pH-Dependent Formation of iM Structures

Zeraati et al. examined how the formation of iM structures is affected by pH in the following way. As depicted here, H2O and CO2 react to form carbonic acid, H2CO3. Consequently, intracellular pH is inversely correlated with the concentration of CO2 in a cell culture system.

Taken from

These investigators hypothesized that they could study the effect of pH on iM formation by changing the CO2 concentration. MCF7 cell cultures were incubated at 5% CO2 until they reached ~70% confluency and then placed in 2%, 5% or 8% CO2 incubators for a further 2.5 h. From the box-and-whiskers plot shown here, staining with iMab led to observation of a relatively small but statistically significant increase in the numbers of foci with increasing CO2 concentration. As expected, measurements of pH in the MCF7 culture medium indicated an inverse correlation with the CO2 concentration. In view of the relatively small lowering of pH, it seems to me that iM structures are quite sensitive to pH changes in cell culture solution.

iMab foci of 200–300 nuclei were counted per condition. Green boxes represent 25th to 75th percentiles. Horizontal lines and “+” indicate medians and means, respectively. Whiskers indicate highest and lowest values of the results. Statistical significance **P < 0.01, ****P < 0.0001. Taken from Zeraati et al. Nature Chemistry (2018)

Cell-Cycle-Dependent Formation of iM Structures

As depicted here, the human cell cycle consists of four distinct phases: G1 phase (growth; cells increase in size), S phase (synthesis; DNA replication), G2 phase (growth and preparation for mitosis, i.e. cell division) and M phase (mitosis). M phase is composed of two tightly coupled processes: karyokinesis, in which the cell’s chromosomes are divided, and cytokinesis, in which the cell’s cytoplasm divides forming two daughter cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase.

Taken from                                     Taken from

To investigate whether iM formation is affected by cell-cycle progression, Zerati et al. used the iMab antibody to stain HeLa cells synchronized at G0/G1, G1/S boundary or early S phase. As shown here, they observed the minimum number of foci at the G0/G1 phase and the highest number of foci at G1/S boundary phase. When the cells synchronously began (early) S phase, the number of iMab foci was reduced compared to the G1/S boundary phase. Similar trends of iM formation during cell-cycle progression were observed in the MCF7 cell line.

Taken from Zeraati et al. Nature Chemistry (2018)

iMab foci of 200–300 nuclei were counted per condition. Green boxes represent 25th to 75th percentiles. Horizontal lines and “+” indicate medians and means, respectively. Whiskers indicate highest and lowest values of the results. Statistical significance **P < 0.01, ****P < 0.0001.


Rather than paraphrase Zeraati et al., here are some salient statements taken from the discussion section:

  • “[W]e were able to detect iMs within a pH range of 6–8. Our results thus indicate that iM DNA structures can indeed form under physiological conditions….”
  • “[T]he formation of iM structures is highly cell-cycle specific…we observed that the highest level of iM formation occurs in late G1 phase, which is characterized by high levels of transcription and cellular growth…iMs may act as scaffolds for the binding of transcription factors during transcription.” 
  • “This behavior is markedly different to GQ formation, which occurs predominately during the S phase. In contrast, we observed a reduction in the number of iMab stained foci in early S phase, indicating that iM structures are resolved during replication.” 
  • “The difference between iM and GQ formation during cell-cycle progression agrees with recent findings demonstrating that these structures are often mutually exclusive and play opposite roles in the regulation of gene expression.”

As mentioned in the introduction, Sir Shankar kindly provided these perspectives on the report by Zeraati et al.:

“[It] is a nice study that adds to the emerging view that DNA secondary structure is more interesting and dynamic than was thought a few decades back.  The results of the study would seem to counter the view that the pH-dependence iMs makes them unlikely to form.  Previous studies have suggested iMs may play roles that are distinct from those proposed for GQs and there is a subtle indication of this from the differences observed in the cell-cycle dynamics of iM, which seem to be most abundant at M1/S and not S-phase, as compared with GQs that max out during S-phase.  As next steps it will interesting to further probe when and where iMs form in the genome and the potential links between iM and function.”  

I thank Sir Shankar for his willingness to share his thoughts here and, as usual, I welcome your comments.


While researching literature for this blog, I became curious about evidence for coexistence of GQ and iM structures in the same region of DNA. One such publication by Chen et al. reports the following:

A diagram of equilibrium between two forms of NHE III1 (nuclease hypersensitivity element III). The left state represents a transcriptionally active form, which can regulate 80-90% of c-Myc transcription, and the right state represents a silenced form, with both GQ and iM structures being shown, which represses the transcription of c-Myc. CNBP: cellular nucleic acid binding protein; hnRNP: heterogeneous nuclear ribonucleoprotein; TBP: TATA-box-binding protein; RNA Pol II: RNA polymerase II. Taken from Chen et al. Int J Biol Sci (2014)

CRISPR in the Clinic…Coming Soon

  • Trio of CRISPR Discoverers Awarded a $1 Million Kavli Prize
  • CRISPR Therapeutics, a Startup Company, Will Soon Start Clinical Trials
  • New Issue: Concerns for Cancer

Over the past few years, I have periodically blogged about CRISPR-based gene editing, which has been arguably the hottest trending topic in nucleic acid-targeted therapy for about the past five years or so. The catalyst for this burst of publications was a 2012 report in Science on a study led by Doudna and Charpentier (see below). The study focused on the potential utility of CRISPR-Cas9 for genome editing, and it currently has over 5000 citations in Google Scholar. There are ~7400 articles in PubMed indexed to CRISPR, and it is evident from my chart shown here that there is strong growth in the annual number of CRISPR publications in the PubMed database.

Number of CRISPR publications in PubMed

In May 2018, three pioneers in CRISPR technology—Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin, Virginijus Šikšnys (see Footnote) of Vilnius University in Lithuania, and Jennifer Doudna of the University of California, Berkeley—were awarded the $1 million Kavli Prize in Nanomedicine. This highly prestigious prize from The Norwegian Academy of Science and Letters was awarded “for the invention of CRISPR-Cas9, a precise nanotool for editing DNA, causing a revolution in biology, agriculture, and medicine.”

Emmanuelle Charpentier, Virginijus Šikšnys and Jennifer Doudna (left to right). Taken from

As far as invention goes, there has been continued litigation, recently summarized in a GEN interview with law professor Jacob Sherkow titled CRISPR in the Courthouse. The University of California, Berkeley (UC) and the Broad Institute of MIT and Harvard are at odds over foundational patents covering CRISPR-Cas9. Interested readers should consult “late breaking news,” which covers the recent issuance of a US patent to UC and its partners.

Notwithstanding unresolved intellectual property matters, so-called “surrogate companies” for the holders of these key patents include Editas Medicine (MIT/Harvard), Caribou Biosciences/Intellia Therapeutics (UC and University of Vienna), and CRISPR Therapeutics (Emmanuelle Charpentier), the latter of which is the focus of the present blog. As you’ll read below, CRISPR Therapeutics is developing a “CTX001” approach for the treatment of Sickle cell disease and β-thalassemia for clinical trials this year, which is a highly anticipated milestone—both scientifically and commercially.

CRISPR Therapeutics CTX001

Sickle cell disease and β-thalassemia are caused by genetic mutations in the β-globin gene, which codes for the β subunit of hemoglobin that, as depicted below, is the oxygen carrying component of red blood cells. In these diseases, hemoglobin is missing or defective, which results in devastating medical problems. The approach developed by CRISPR Therapeutics is designed to mimic the presence of fetal hemoglobin (HbF; aka γ-globin) that is present in newborn babies. HbF is a form of hemoglobin that is quickly replaced by adult hemoglobin. However, in rare cases where HbF persists in adults, it provides a protective effect for those who have Sickle cell disease and β-thalassemia.

Taken from

CTX001 is an ex vivo therapy in which autologous (i.e. self-donated) cells are harvested directly from the patient. CRISPR Therapeutics then applies its gene-editing technology to the cells outside of the body, making a single genetic change designed to increase HbF levels in a patient’s own blood cells. The edited cells are then reinfused and are expected to produce red blood cells that contain HbF in the patient’s body, thus overcoming the hemoglobin deficiencies caused by these diseases.

The gene-editing mechanism for CTX001 presented by CRISPR Therapeutics at the American Society of Hematology (ASH) in December 2017 is depicted below. In researching this CRISPR-based mechanism, I found a publication by Bjurström et al. that helps to better understand this depiction. In brief, the zinc-finger transcriptional factor BCL11A has been shown to silence HbF genes in human cells during development, and thus directly regulates HbF switching.

Taken from CRISPR Therapeutics

BCL11A silences HbF by associating with other known γ-globin transcriptional repressors. The gene binds to the locus control region as well as other intergenic sites, which prevents the interaction between the locus control region and the HbF globin gene required for fetal globin expression. Using a guide RNA and Cas9 to enable permanent site-specific genome engineering through a DNA repair pathway, knockdown of the BCL11A gene can be an effective strategy for reactivating HbF and restoring functional erythrocytes.

The aforementioned ASH presentation by CRISPR Therapeutics also includes an overview of Sickle cell disease and β-thalassemia, as shown here. According to an informative historical article that I found, Sickle cell disease and β-thalassemia are related genetic disorders that can cause fatigue, jaundice, and episodes of pain ranging from mild to very severe. They are inherited, and usually both parents must pass on an abnormal gene in order for a child to have the disease. Much more genetic information on these two disorders is available on the NIH Genetics Home Reference.

Taken from CRISPR Therapeutics

CRISPR Therapeutics Clinical Studies Status

The December 2017 ASH presentation by CRISPR Therapeutics received widespread media coverage that heralded the highly anticipated “bench-to-bedside” transition for CRISPR technology. CTX001 was able to efficiently edit the target gene in more than 90 percent of hematopoietic stem cells to achieve about 40 percent of HbF production, which investigators believe is sufficient to improve a patient’s symptoms. Study results also showed that CTX001 affects only cells at the target site and that it has no off-target effects on hematopoietic stem cells, thereby appearing to be a safe potential treatment.

These positive results prompted CRISPR Therapeutics to start a collaboration with Vertex Pharmaceuticals to develop and commercialize CTX001 treatment of Sickle cell disease and β-thalassemia. It was also announced that CRISPR Therapeutics and Vertex are planning to submit an investigational new drug (IND) application to the Food and Drug Administration (FDA) to start a Phase 1/2 clinical trial in Sickle cell disease in the United States in 2018. In addition, CRISPR Therapeutics also submitted a clinical trial application (CTA) for CTX001 to advance into a Phase 1/2 clinical trial in patients with β-thalassemia in Europe in 2018. This trial will evaluate the safety and effectiveness of CTX001 in adult patients with transfusion-dependent β-thalassemia.

After the above announcement, News Atlas reported that the FDA placed a clinical hold on this Phase1/2 trial of CTX001 pending, according to CRISPR Therapeutics, ‘the resolution of certain questions that will be provided by the FDA as part of its review of the IND.’

Concerns for Cancer

In studies published in June 2018 in venerable Nature Medicine, researchers from Sweden’s Karolinska Institute and, separately, Novartis, found that cells whose genomes are successfully edited by CRISPR-Cas9 have the potential to seed tumors inside a patient. CRISPR-Cas9 works by cutting both strands of the DNA double helix. That “injury” causes a cell to activate a gene called p53, which has been called the “Guardian Angel of the Genome” and is the most studied of all human genes, which you can read about in one of my previous blogs.

Whichever action p53 takes, the consequence is the same: CRISPR doesn’t work as intended because the genome edit is mended, or the cell dies. The flip-side of p53 repairing CRISPR edits, or killing cells that accept the edits, is that cells that survive with the edits do so because they have a dysfunctional p53. The reason why that could be a problem is that p53 dysfunction can cause cancer. The p53 gene is reported to be the most frequently mutated gene in human cancer: about 50% of all human cancers have lost p53 or express an inactive, mutant p53.

As a result, the Novartis paper concludes that “it will be critical to ensure that [genome-edited cells] have a functional p53 before and after [genome] engineering.” The Karolinska team warns that p53 and related genes “should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.”

An article in quotes the CEO of CRISPR Therapeutics, Sam Kulkarni, as saying that these p53 findings are “something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous.”

Closing Comments

Taken from

Many years ago, I was among the early investigators of antisense therapeutics, which at the time was viewed as a new paradigm that would enable faster bench-to bedside, compared to traditional small molecule drug development. In reality, the antisense approach encountered unforeseen complications and required ~30 years of development to reach demonstrable clinical utility, which I previously wrote about in another blog. Short-interfering RNA (siRNA)-based therapeutics also encountered similar struggles.

While past history is not a predictor of the future, in my humble opinion, CRISPR-based clinical strategies will continue to have to deal with unexpected issues, such as the above p53 situation. While I remain hopefully optimistic about future clinical successes for CRISPR, I won’t be surprised if some of these achievements come slower than currently anticipated.

As usual, your comments are welcomed.


According to June 8, 2018 Science News at a Glance, Virginijus Šikšnys, whose role in the invention of the revolutionary genome editor CRISPR has often been overlooked, received some vindication when he was named a co-winner of the prestigious Kavli Prize in Nanoscience. Šikšnys will share the $1 million award with Doudna and Charpentier, who have received far more attention. Šikšnys first showed that the CRISPR system could be transferred from one bacterium to another. And like Doudna and Charpentier, he independently designed a way to steer the CRISPR complex to specific targets on a genome, which he called “directed DNA surgery.”




ATP Synthase and ATPase: In Memory of Paul D. Boyer and Jens C. Skou

  • ATP Is the “Molecular Unit of Currency” of Intracellular Energy Transfer
  • 1997 Nobelists Boyer and Skou Elucidated Fundamental ATP Biochemistry
  • Both Laureates Have Recently Passed Away at Age 99

In 1977, the Nobel Prize in Chemistry was divided among three scientists for their fundamental contributions related to adenosine triphosphate (ATP), which is often referred to as the “molecular unit of currency” of intracellular energy transfer. One half of this prize was jointly awarded to Paul D. Boyer and John E. Walker for their elucidation of the enzymatic mechanism underlying the synthesis of ATP, and the other half was awarded to Jens C. Skou for the first discovery of an ATP-driven ion-transporting enzyme: sodium/potassium-ATPase.

Taken from

ATP sodium. Taken from TriLink BioTechnologies

The recent passing of Boyer (June 2, 2018) and Skou (May 28, 2018) is doubly saddening, as well as striking in that both Nobelists died at age 99, and within only five days of each other. In memory of their fundamentally important contributions related to ATP, which is arguably the most important nucleotide for life, this blog provides a brief synopsis of the amazing mechanism by which ATP is produced by ATP synthase, and the equally amazing mechanism by which ATP pumps sodium and potassium ions across cell membranes.

ATP Synthase

I highly recommend reading Boyer’s historical account and personal perspective on how, alongside his graduate students and postdoctoral fellows, over a span of many years, he concluded that ATP is made by an unusual rotational catalysis of ATP synthase. Boyer’s reflections also explain how an x-ray structure reported by Walker—now an emeritus director of the MRC Mitochondrial Biology Unit in Cambridge—came to play a critical role in establishing this novel mechanism.

Taken from Boyer J Biol Chem (2002)

There are three major conformations of catalytic sites (1, 2 and 3) for synthesis of ATP or hydrolysis by ATP. These three sites are depicted here with asymmetric interactions to the central subunit. During catalysis, sites are converted sequentially into three different states, accompanying rotation of the central subunit. The sequence for synthesis is 1 → 2 → 3, while for hydrolysis it is 3 → 2 → 1. Site 1 binds adenosine diphosphate (ADP) better than ATP and is the site at which ADP and inorganic phosphate (Pi) must be present for rapid synthesis to occur. Site 2 has the ability to catalyze chemical transformation and to be present as a form with ADP and Pi present or with ATP present. ATP can be released from Site 3 during synthesis and must be present at this site for rapid hydrolysis.

The rotation of the central subunit is powered, so to speak, by protons. To fully appreciate the wonderous complexity of this “molecular machine”, it’s necessary to consider all of the structural components and the functions ascribed to each. The colorized depiction of membrane-imbedded ATP synthase from E. coli shown below is a useful visual aid for such mechanistic considerations.

Taken from Boyer J Biol Chem (2002)

This ATP synthase consists of two regions called F1 and F0. The F1 portion subunits are designated as α3β3γδε and the F0 portion subunits are designated as ab2c9-12. The passage of protons (H+), at the interface of subunit a and the ring of c subunits, causes a rotation of the c subunits and attached ε and γ subunits relative to the rest of the enzyme. The asymmetric γ subunit (yellow and light green) extends through the center of the α3β3 cluster. The b2 and δ subunits serve as a stator, i.e. the stationary part of a rotary system. The rotation of the γ subunit results in sequential conformational changes of the catalytic sites that promote ADP and Pi binding, ATP formation, and ATP release. You can access and read a concise recap of this process written by Boyer at this link in a Nature article titled What makes ATP synthase spin?

Amazing ATP Facts

I was truly amazed—and totally surprised—by the following facts on ATP, especially the last bullet point:

  • Cellular ATP concentration is normally maintained in the range of 1 to 10 mmol/L, with a normal ratio of ATP/ADP of approximately 1000.
  • The totally quantity of ATP in an adult is approximately 0.10 mol/L.
  • Approximately 100 to 150 mol/L of ATP are required daily, which means that each ATP molecule is recycled some 1000 to 1500 times per day.
  • Transmembrane proton flux through the mitochondrial ATPase synthase complex occurs at an estimated rate of 3 × 1021 protons per second.
  • This corresponds to ATP reformed at a rate of 9 × 1020 molecules/sec, or approximately 65 kg ATP recycled per day in a normal resting adult.
  • Basically, the human body recycles its weight in ATP daily!

Sodium/Potassium-ATPase (Na/K-ATPase)

Taken from

To fully appreciate the scientific story regarding the first discovery of Na/K-ATPase, I recommend reading Skou’s historical account of the circumstances that led him to this achievement. Skou recalls reading a paper that mentioned the presence of an ATP hydrolyzing enzyme in the sheath part of the giant axon of a squid: an ATPase. Knowing then that ATP is the energy source within cells, Skou was intrigued, but he had no access to giant axons and therefore no way of investigating this putative nerve membrane ATPase. He reasoned that crab nerves might be a suitable alternative, so in 1954, he made a deal with a local fisherman to provide him with crabs. He isolated the sciatic nerve from the legs, starting what would become his pioneering discovery.

By 1965, Skou had accumulated enough data to publish a review, summarizing the following properties for the enzyme he named “Na+, K+-ATPase”:

  • It is located in the cell membrane.
  • It has a higher affinity for Na+ than for K+ on the cytoplasmic side; and a higher affinity for K+ than Na+ on the extracellular side.
  • It has enzymatic activity and catalyzes ATP hydrolysis; the rate of which depends on cytoplasmic Na+ and extracellular K+.
  • It is found in all cells that have coupled active transport of Na+ and K+.

The simplified series of depictions shown below represent the current view of the mechanistic steps for how Na/K-ATPase functions as a sodium-potassium pump. (1) The Na/K pump binds three sodium ions and a molecule of ATP. (2) The splitting of ATP into bound Pi and ADP provides energy to change the shape of the channel, which leads to sodium ions being driven through the channel. (3) The sodium ions are released to the outside of the membrane, and the new shape of the channel allows two potassium ions to bind. (4) Release of Pi allows the channel to revert to its original form, releasing the potassium ions on the inside of the membrane.

Taken from

Legacy of Literature

In my humble opinion, one of the most gratifying aspects of being a scientist is recognizing that your published investigations will be available for others to learn from after you are no longer among the living. This immortalization, if you will, is what we can collectively think of as our “legacy of literature” that can benefit, in some way, future generations of scientists and other members of society.

The Nobel-worthy contributions by Boyer and Skou on the elucidation of ATP biochemistry are obviously very special examples of this legacy, as reflected in the huge numbers of citations to their publications. I used Google Scholar to find several of the most highly cited articles by each of these Nobelists that you can peruse by clicking the link to each:

Citations          Title

2513               The influence of some cations on an adenosine triphosphatase from peripheral nerves

2378                 Enzymatic Basis for Active Transport of Na+ and K+ Across Cell Membrane

1823                 The ATP synthase—a splendid molecular machine

1157                 The binding change mechanism for ATP synthase—some probabilities and possibilities

873                    Further investigations on a Mg+++ Na+-activated adenosine triphosphatase…

700                     Oxidative phosphorylation and photophosphorylation

By the same token, Walker’s ATP-related legacy of literature can be perused via this link to all of his publications in PubMed. In researching the literature for background and details, I learned a lot about ATP synthase and ATPase biochemistry. Hopefully, you have also found some of this information new and interesting.

Finally, I came across this quote by Paul Boyer expressing his thoughts about being awarded the Nobel Prize after many years of slogging through seemingly endless enzymology; it resonated with me and may also resonate with you:

“The experience reminds me of a favorite saying: Most of the yield from research efforts comes from the coal that is mined while looking for diamonds.”

Taken from

As usual, your comments are welcomed.

June Is Acute Myeloid Leukemia (AML) Awareness Month

  • N6 Methylation of Adenosine in mRNA by METTL3 Linked to AML
  • Linking METTL3 to AML by Gene Knockdown with RNA Interference
  • Targeting METTL3 May Lead to a New Drug for AML

Designation of June as Acute Myeloid Leukemia (AML) Month was announced in 2016 by CancerCare®, which provides free emotional and practical support for people with cancer, caregivers, loved ones and the bereaved. The stated goal of the campaign is to put a spotlight on this rare and difficult-to-treat blood cancer that typically affects older adults. In support of that goal, the present blog focuses on newly reported mechanistic findings related to AML.

Taken from

By way of an introduction, previous blogs here on RNA epigenetics dealt with discovery of post-transcriptionally modified bases in RNA and enzymes that add (“write”) and remove (“erase”) these modifications, and the effects on RNA function. Among these base-modified RNAs, N6-methyladenosine (m6A; shown here), which is present in bacterial and eukaryotic cells, has been found to have a regulatory role in RNA processing.

According to Wei et al., m6A occurs mainly in the 3′ untranslated regions (UTRs) and near the stop codons of mRNA. Dynamic regulation (“editing”) of m6A is found in metabolism, embryogenesis, and developmental processes, indicating an epigenetic regulatory role. For example, it is known that m6A editing is involved in nuclear RNA export, mRNA degradation, protein translation, and RNA splicing. More recently, as summarized below, m6A editing has been linked to human cancer.

m6A Formation in mRNA Mediated by METTL3 Linked to AML

Although m6A is required for differentiation of mouse embryonic stem cells, it has been unknown whether m6A controls differentiation of normal and/or malignant myeloid hematopoietic cells. Compelling evidence in support of such control by m6A was recently published by Vu et al. in Nature Medicine based on collaborative work by a large team of investigators at Memorial Sloan Kettering Cancer Center (NY), Weill-Cornell Medical College (NY), and elsewhere. Readers interested in details should consult this lengthy paper, but for now here’s my short synopsis of what was done.

By way of background, N6-adenosine-methyltransferase 70-kDa subunit (METTL3) is an enzyme that in humans is encoded by the METTL3 gene and, in complex with an analogous subunit (METTL14), is involved in post-transcriptional methylation of the N6 position of adenosine residues in eukaryotic mRNAs to form m6A. The molecular structure and mechanism of how this happens are published and are schematically depicted in the drawing shown below, taken from that work by Śledź and Jinek.

S-adenosyl methionine (SAM); active site loops (ASLs) 1 and 2. Taken from Śledź & Jinek eLife (2016)

Taken from

Since myeloid differentiation (see below) is frequently dysregulated in leukemia, Vu et al. determined if METTL3 expression is altered in leukemia. METTL3 mRNA expression in human AML samples is significantly higher than in other cancer types. To further assess the relative abundance of METTL3 in myeloid leukemia, they examined METTL3 mRNA and protein levels in 11 multiple leukemia cell lines and compared these to primary human cord blood-derived CD34+ cells. METTL3 mRNA was more abundant in AML cell lines (8/11), as was METTL3 protein (11/11). They found no significant difference in METTL3 expression across multiple subtypes of AML.

Taken from

After finding increased METTL3 in leukemic cells versus normal cells, they next measured m6A abundance in mRNA and found a significant increase in an AML cell line (MOLM13) compared to CD34+ control cells. These data suggested that elevated m6A might be critical to maintaining an undifferentiated state in myeloid leukemia. To directly address the role of m6A in human myeloid leukemia cells, they examined the effect of METTL3 mRNA knockdown in MOLM13 cells using short hairpin RNA (shRNA). METTL3 knockdown significantly decreased m6A levels, blocked cell growth, induced differentiation and resulted in an increase in apoptosis (aka “programmed cell death”). Similar effects of shRNA-mediated METTL3 depletion were seen in two other AML cell lines.

Vu et al. next investigated whether METTL3 was required in vivo for induction of leukemia. MOLM13 cells were transduced with METTL3-shRNA and transplanted into immunodeficient recipient mice. These cells exhibited delayed leukemia development compared to MOLM13 cells transduced with the scrambled-shRNA. To determine how METTL3 depletion affects m6A -containing transcripts, they performed RNA-Seq on MOLM13 cells following METTL3 knockdown. Consistent with the idea that m6A destabilizes mRNA, transcripts with at least one m6A site showed increased abundance following METTL3 depletion compared to transcripts with no called m6A sites. Moreover, the change in abundance was directly correlated with the number of m6A sites per transcript.

Clinical Relevance

To assess clinical relevance of the MOLM13 m6A profile, Vu et al. mapped m6A in two AML patient samples and compared these transcripts to m6A-enriched transcripts found in MOLM13 cells. They found that the mRNAs with the highest levels of m6A (top 300 transcripts) from the patient samples were enriched with the m6A transcripts from the MOLM13 cells.

Vu et al. stated that this study is the first to demonstrate that m6A is critical for maintaining the differentiation program in the hematopoietic system and that this process is dysregulated in myeloid leukemia. Rather than rephrasing the final conclusion by these researchers, here’s the direct quote:

Our data suggest that inhibition of METTL3 could be exploited as a therapeutic strategy for myeloid malignancies. It is notable that leukemia cells show elevated abundance of METTL3 compared to normal hematopoietic cells. Furthermore, we find that METTL3 depletion shows markedly increased levels of apoptosis in leukemia cells compared to normal cells. These findings suggest a possible therapeutic index. Future studies that target METTL3, potentially in combination with current and emerging therapeutic agents, should be explored.

Parting Comments

Given the increasing interest in elucidation of how mRNAs bearing various types of base-modifications are related to cancer, it seems likely that more discoveries will lead to future therapies of the sort envisaged by Vu et al. for m6A in AML.

Finally, in recognition of June as AML Awareness Month, I thought it would be worth sharing the following information about this disease taken from an authoritative website maintained by the National Cancer Institute.

  • The number of new cases of AML was 4.3 per 100,000 people per year, while the number of deaths was 2.8 per 100,000 people per year.
  • Lifetime risk of developing AML is ~5% of people will be diagnosed with AML at some point during their lifetime, based on 2013-2015 data.
  • Less than 30% of people survive5 years or more after being diagnosed with AML.
  • AML is most frequently diagnosed among older people but it effects all age groups.
  • Leukemia is cancer that starts in the tissue that forms blood. Most blood cells develop from cells in the bone marrow called stem cells. In a person with leukemia, the bone marrow makes abnormal white blood cells. The abnormal cells are leukemia cells. Unlike normal blood cells, leukemia cells don’t die when they should. They may crowd out normal white blood cells, red blood cells, and platelets. This makes it hard for normal blood cells to do their work.
  • There are four main types of leukemia: AML, acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML).

Common symptoms for leukemia are as follows:

Taken from

As usual, your comments are welcomed.







Using Nucleotide Analogs for RNA Visualization and Gene Expression Kinetics

  • 5-Azidoalkyluridine in RNA for Click Chemistry in Living Cells for Visualization
  • 4-Thiouridine in RNA in Living Cells for Kinetics by Sequencing

Homeostasis is defined as the stable state (or equilibrium) of an organism and of its internal environment. Maintenance and regulation of homeostasis involves regulated expression of genetic information, and disruption of this process can result in human diseases. The transient existence and function of RNA in the framework of the central dogma of molecular biology necessitates tightly regulated molecular events. In turn, these events control the relative kinetics of RNA transcription, processing, and degradation. To understand the molecular basis for gene regulation, we first must understand the relative kinetics of RNAs biogenesis and degradation in a systematic and transcript-specific manner.

Taken from

Readers interested in learning about the details concerning the biogenesis and degradation of RNA can consult a comprehensive survey and summary by Jackowiak et al., which also serves as a source of references for numerous subtopics. This “chemistry-centric” blog will first focus on fluorescence-based visualization of RNA using click chemistry, which I’ve blogged about previously. Following that, there will be a synopsis of how the chemical properties of the nucleotide analog 4-thiouridine enable high-throughput, sequencing-based RNA, counting for determination of gene expression kinetics.

RNA Visualization

RNA visualization methods commonly rely on metabolic labeling of RNA with ribonucleoside or ribonucleotide analogs, such as 5-bromouridine (BrU) or BrU-5’-triphosphate, followed by immunostaining with fluorescent antibody for BrU, as described here. However, these methods involve laborious assay setups and are not applicable to all cell types and tissue samples due to limited permeability of the antibodies. Endogenous RNA has also been visualized by using fluorescently-modified antisense oligonucleotide probes, molecular beacons, and, more recently, aptamer-binding fluorophores. These methods have varying benefits and limitations, which have been reviewed by Mannack et al. in a freely accessible publication in 2016 titled Current techniques for visualizing RNA in cells that is definitely worth reading as an introduction to this field.

Taken from Mannack et al. F1000Res (2016)

Sawant et al. describe the development of a versatile toolbox composed of azide-modified uridine triphosphates, which facilitates the direct incorporation of azide functionality into RNA transcripts by transcription reaction, as depicted here. The azide-modified RNA is readily functionalized with biophysical probes by various types of reactions, including strain-promoted azide-alkyne cycloaddition (SPAAC) and azide-phosphine Staudinger chemical ligation. Importantly, Sawant et al. show the specific incorporation of azide groups into cellular RNA transcripts by endogenous RNA polymerases for the first time. The azide-labeled cellular RNA transcripts are conveniently visualized in fixed and live cells by fluorescence microscopy upon click reaction with fluorescent alkynes.

Taken from Sawant et al. Nucleic Acids Res (2015)

Interested readers can consult Sawant et al. for extensive details regarding synthesis of the azido-functionalized nucleoside triphosphates and fluorescently labeled reagents that have ring-strained reactive carbon-carbon triple bonds, such as the Cy3-labeled cyclooctyne example shown here.

Taken from Sawant et al. Nucleic Acids Res (2015)

The utility of the SPAAC reaction in the detection of newly transcribing RNA in living cells was first observed by treating the cells with 5-azidomethyluridine-5’-triphosphate (AMUTP) for 1h. Subsequently, the cells were treated with a Cy3-labeled cyclooctyne probe for 30min and washed. The resultant Cy3 SPAAC RNA product was observed by confocal microscopy. Cell viability tests were done to confirm the low toxicity of the probe in these staining reaction conditions. According to Sawant et al., this RNA labeling method is advantageous, as the selective incorporation of azide groups into RNA using AMUTP and the SPAAC dye-visualization procedure provide an alternative route to image actively transcribing RNA in living cells.

Gene Expression Dynamics

Metabolic RNA labeling approaches that employ nucleotide-analogs enable tracking of RNA species over time without interfering with cellular integrity. Among these, 4-thiouridine (s4U) represents the most widely used nucleotide-analog to study the dynamics of RNA expression. This is because it is readily imported into metazoan cells by equilibrate nucleoside transporters, and it provides unique physicochemical properties for thiol(SH)-specific reactivity.

Herzog et al. recently reported SH-linked alkylation for the metabolic sequencing (SLAM-seq) of RNA to precisely locate s4U at single-nucleotide-resolution by reverse-transcription-dependent thymine-to-cytosine(T>C)-conversions in a high-throughput sequencing-compatible manner. For SLAM-seq, Herzog et al. employed the SH-reactive compound iodoacetamide, which covalently attaches a carboxyamidomethyl-group to s4U by nucleophilic substitution, as depicted here.

Taken from Herzog et al. Nature Methods (2017)

Quantitative s4U-alkylation was confirmed by a shift in the characteristic absorbance spectrum of 4-thiouracil from 335 nm to 297 nm. Under optimal reaction conditions, absorbance at 335 nm decreased 50-fold compared to untreated 4-thiouracil, resulting in ≥98% alkylation within 15 min. Quantitative identification of s4U by sequencing presumes that reverse transcriptase (RT) passes alkylated s4U-residues without drop-off. Herzog et al. therefore studied the effect of s4U-alkylation on RT-processivity in primer extension assays, but did not observe a significant effect of s4U alkylation on RT processivity when compared to a non-s4U-containing oligo with identical sequence.

To evaluate the effect of s4U-alkylation on RT-directed nucleotide incorporation, these investigators isolated the full-length products of primer extension reactions, PCR-amplified the cDNA, and subjected the libraries to high-throughput-sequencing. While the presence of s4U led to a constant ~10% T>C-conversions in the absence of alkylation (presumably due to base-pairing variations of s4U-tautomers), s4U-alkylation increased T>C-conversions 8.5-fold, resulting in a >94% T>C conversion rate. Importantly, they showed that iodoacetamide-treatment leaves conversion rates of any given non-thiol-containing nucleotide unaltered. Interested readers can consult the publication for details on how sequencing data are processed to give T>C conversion results.

The overall workflow of SLAM-seq is depicted below. To directly measure mRNA transcript stabilities, Herzog et al. subjected mouse embryonic stem cells (mESCs) to s4U metabolic RNA labeling for 24h, followed by washout and chase using uridine. They also prepared total RNA at various time points along the chase, which was then subjected to alkylation and Quant-seq. Inspection of candidate genes revealed constant steady-state expression across the time-course, while T>C-conversion-containing reads decreased over time in a transcript-specific manner. After suitable calculations, normalized T>C-conversion rates fit well to single-exponential decay kinetics, enabling the determination of transcript half-life.

Workflow of SLAM-seq. Working time for alkylation and Quant-seq library preparation are indicated. Taken from Herzog et al. Nature Methods (2017)

Herzog et al. ranked the 6,665 transcripts for which half-life was determined at high accuracy according to their relative stability. They also performed gene-ontology (GO) enrichment analysis for the 666 most or least stable mRNAs, as shown here. Transcripts with short half-life significantly enriched for regulators of Pol II-dependent transcription, while stable mRNAs associated with the GO-terms translation, respiratory electron transport, and oxidative phosphorylation. Together with gene set enrichment analyses, SLAM-seq measurements confirmed that transcripts encoding proteins with house-keeping function tend to decay at low rates, perhaps reflecting the evolutionary adaptation to energy constraints. In contrast, transcripts with a regulatory role tend to decay faster, most certainly because control over the persistence of genetic information facilitates adaptation to environmental changes.

Taken from Herzog et al. Nature Methods (2017)

Herzog et al. point out a number of limitations and caveats for SLAM-seq RNA kinetics. Because s4U-uptake can vary between cell types, careful assessment of cell-type-specific toxicity is imperative in order to meet s4U-labeling conditions that do not affect gene expression or cell viability. Additionally, the ability to determine de novo synthesized transcripts will depend on (1) the cellular s4U uptake kinetics, (2) the overall transcriptional activity of the cell type and (3) the library sequencing depth. Hence, these parameters need to be taken into account when designing a SLAM-seq experiment, particularly when employing short s4U pulse labeling, where sequencing depth demands adjustments to the given cellular parameters.

Notwithstanding these issues and reflecting on what SLAM-seq has revealed about gene expression kinetics, I was struck by the remarkable effects derived from simply substituting a sulfur atom for oxygen in uridine. Chemistry is amazing!

As usual, your comments are welcomed.


Reading about s4U piqued my curiosity about publications in which s4U, as the nucleoside or triphosphate obtained from TriLink, has been used. I searched Google Scholar for such publications, and found a relatively large number and variety of applications, which you can peruse here. My favorite was a protocol by Timothy W. Nilsen in RNA: A Laboratory Manual titled Detecting RNA–protein interactions by photocross-linking using RNA molecules containing uridine analogs.




FDA Approves First-of-a-Kind Test for Profiling Cancer Genes

  • Memorial Sloan Kettering Develops a Next-Generation Sequencing (NGS) Assay for 468 Genes
  • FoundationOne Does the Same for a 324 Gene Assay
  • Some View These Approvals as Tearing Down Conceptual Walls Between System Biology and Clinical Practices

Taken from (Credit: C. Lynm)

This blog’s title, which recently flashed around the globe as headline news, deserves to be echoed here, as it signals the achievement of a major milestone in nucleic acid-based diagnostics. Ever since the emergence of powerful PCR and sequencing methods for DNA analysis, there have been thousands of publications dealing with discovery and validation of genes associated with the genesis or signature of various types of cancer. A great deal of literature has also appeared regarding possible use of DNA analysis for cancer diagnostics.

Despite these continuing advances, the transition from research findings to doctors using DNA diagnostics has been hindered by the relatively long and costly process of gaining approval by regulatory authorities. In the U.S., the regulatory authority is the Food and Drug Administration (FDA). Readers interested in regulatory aspects can use this link to access FDA guidance for genetic testing in general. The focus herein is to provide a brief overview of what underlies the headline news echoed in the title of this blog.

Mutational Landscape of Metastatic Cancer

Molecular pathology of cancer has historically relied upon low-throughput approaches to interrogate a single allele in a single sample, such as those listed at this FDA website. By contrast, massively parallel “next generation” sequencing (NGS) has enabled a dramatic expansion in the content and throughput of diagnostic testing. However, the complexity of clinical NGS testing has prevented laboratories from achieving large-scale implementation, which is needed in order to maximize the benefits of tumor genomic profiling for large populations of patients. In addition, the clinical utility of mutation profiling requires evaluation of how molecular results are influencing therapeutic decisions in different clinical contexts.

To address this issue, the Memorial Sloan Kettering (MSK) Cancer Center developed a targeted tumor sequencing test, MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), to detect gene mutations and other critical genetic aberrations in both rare and common cancers. This test therefore detects all protein-coding mutations, copy number alterations, selected promoter mutations, and structural rearrangements in 341 (and more recently, 468) cancer-associated genes, as detailed by Zehir et al. This large team of investigators prospectively sequenced tumors from more than 10,000 cancer patients, who presented with a vast array of solid tumor types.

Taken Zehir et al. Nature Medicine (2017)

The DNA sample prep and analysis methodology described by Zehir et al should be appreciated as a tour de force of integrated automated sample handling systems. The method operates in conjunction with barcoded adapters, PCR, multiplexed DNA capture by biotinylated hybridization probes, and paired-end 100-base pair NGS reads – all to a mean depth of coverage of 718X.

Breakthrough FDA Approvals

On November 15th 2017, the FDA announced approval of MSK-IMPACT as a tumor profiling assay, i.e. an in vitro diagnostic (IVD) test, which can identify a higher number of genetic cancer mutations (biomarkers) than any test previously reviewed by the agency. The IMPACT test works by comparing a tumor tissue sample to a “normal” sample of tissue or cells from the same patient. This serves to detect genetic alterations that might help guide treatment options.

While the test is intended to provide information on cancer biomarkers, its results are not conclusive for choosing a corresponding treatment according to the FDA, which added that the assay is >99% accurate and capable of detecting a mutation at a frequency of ~5%. Detection of certain molecular changes (microsatellite instability) using the IMPACT test was concordant >92% of the time across multiple cancer types in 175 cases when compared to traditional methods of detection. Importantly, the Centers for Medicare & Medicaid Services (CMS) cover the cost of this test.

Shortly thereafter, on November 30th, the FDA announced approval of FoundationOne CDx (F1CDx) as an NGS-based IVD test that can detect genetic mutations in 324 genes and two genomic signatures in any solid tumor type. This test will also be covered by CMS. Additionally, based on individual test results, the new diagnostic can identify which patients with any of 5 tumor types may benefit from 15 different FDA-approved targeted treatment options. Its results provide patients and health care professionals all of this information in one test report, avoiding duplicative biopsies.

Tearing Down the Walls

A very recently published commentary by Allegretti et al. argues that, besides the many practice-changing implications, MSK-IMPACT and F1CDx approval by the FDA “tears down the conceptual walls dividing system biology from clinical practice, diagnosis from research, prevention from therapy, cancer genetics from cancer genomics, and computational biology from empirical therapy assignment.”

These authors further opine that that MSK-IMPACT and F1CDx have moral and ethical implications. For the first time, the FDA implies—and, the authors posit, some may say FDA plainly endorses the view—that “each patient at an advanced cancer stage has the right to have her/his cancer genome deciphered at the highest possible level of complexity compatible with current knowledge and technology, linking molecular information to state-of-the-art systemic therapies, as they become available.”

Therefore, according to Allegretti et al., extended NGS testing is becoming the standard of care in oncology. In the near future, “NGS profiling will likely be requested at progressively earlier stages, leading to a change in the engagement rules. No longer will the oncologist request a single assay for a single therapeutic option, e.g. BRAF or RAS mutational status for specific pathway blockade in specific cancers. On the contrary, it is implicit in [these] FDA approvals that the entire mutation catalogue will have to be made available to the medical team as soon as possible after diagnosis, and much before any specific therapy becomes applicable.” Pictorially, they view the new paradigm as follows:

Taken from Allegretti et al. J Exp Clin Cancer Res (2018)

I hope you will forgive my extensive quoting from Allegretti et al., but I believe it’s important to accurately convey the importance of their argument, given the profound impact of this type of thinking. Only time will tell the extent to which change become reality, as these innovations must deal with the hurdle of difficult dynamics between cancer care, cost containment, and societal conscience.

As always, your comments are welcome.



DNA Origami—Self-Assembling DNA Nanostructures

  • Ned Seeman’s Beer-Assisted “Epiphany” for DNA Self-Assembly Has Led to DNA Nanotechnology 
  • Creating DNA Origami Is the DNA-Equivalent of the Japanese Art of Paper Folding 
  • You’ll Be Amazed at What Can Now Be Made from DNA Origami

I first came to know Nadrian “Ned” Seeman by his call-in to me many years ago at Applied Biosystems when he wanted my help in synthesizing highly self-complementary DNA oligos, which can sometimes be challenging. He told me about his interest in using these oligos to study unusual DNA structures related to naturally occurring Holliday junctions. I was intrigued, agreed to help, and we met shortly thereafter at a Gordon Conference on nucleic acids. Little did I know then that Ned’s seemingly quirky-to-me ideas would eventually lead to an entirely new class of self-assembling macromolecules now referred to as DNA nanostructures.

Ned Seeman. Taken from

This video link will take you to Ned Seeman’s personal recollection of how, back then, his drinking beer to better muse about imaginary 6-armed junctions of DNA—instead of Holliday’s 4-armed version—led to Ned’s “epiphany,” in which he envisaged self-assembly of his hypothetical structures to be like Escher’s “Flying Fish,” woodcut print shown here.

Flying Fish, by MC Escher, 1955. Taken from

Taken from

For reasons that you’ll appreciate by reading on, Ned’s use of simple A/T and G/C base pairing (i.e. hybridization) for the design of elaborate self-assembling DNA nanostructures has paved the way for the creation of some amazing constructs involving DNA folding. Because this process is akin to creating origami sculptures by paper folding, the phrase DNA origami has come into usage.

Having introduced you to Ned and his fathering of the field of DNA nanostructures, I think his 1991 publication in venerable Nature magazine titled Synthesis from DNA of a Molecule with the Connectivity of a Cube catapulted his ideas into high visibility by the scientific community. Interested readers should consult this now classic paper for details on how this was achieved, but for now the below model of such a cube, taken from his lab’s visually entertaining website, reveals the overall cubic shape.

Taken from

In this representation of a DNA cube, the DNA backbones are shown in red (front), green (right), yellow (back), magenta (left), cyan (top) and dark blue (bottom). Each nucleotide is represented by a single colored dot for the backbone and a single white dot for the base. Each edge of the cube is a piece of double helical DNA, containing two turns of the double helix. Ned’s 1991 Nature paper reporting “the first construction of a closed polyhedral object from DNA” concludes with the sentence below, which in retrospect is both prescient and an understatement of what would then follow his work:

“The synthesis of this object establishes that it may be feasible to make larger and more complex objects.”


Fast forward nearly 30 years and let me take you to some exemplary “larger and more complex objects” selected from a lengthy, comprehensive 2017 review by Hong et al. titled DNA Origami: Scaffolds for Creating Higher Order Structures. According to this review, Paul Rothemund at California Institute of Technology was the first to report the DNA origami technique in a 2006 paper in Nature. The self-assembly process for a DNA “smiley face” is depicted here.

Taken from Hong et al. Chemical Reviews (2017)

The steps involved are: (A) a target shape is first approximated with parallel cylinders that are (B) tightly aligned in parallel and that (C) utilize DNA helices. (D) An example of a DNA origami scaffold with “staple” strand routing—use your screen zoom to see these better; the dark blue strand is the “scaffold”, and the staple strands are shown in various colors. (E) The scaffold and the staples (with a large excess of the staples) are mixed, and the designed structures self-assemble with a cooling temperature ramp in a salted buffer. (F) The atomic force microscopy (AFM) image of an assembled DNA origami with a smiley face pattern.

Hong et al. say that rather than using a chemically synthesized strand, it is much cheaper and convenient to use a strand of single-stranded DNA from a viral genome as the scaffold because these genomes are thousands of nucleotides long and originate from a bacteriophage virus with known sequences. The genome of the M13mp18 virus is the most widely used scaffold for assembling DNA origami structures. The scaffold and the staple strands are mixed in buffer and slowly annealed from 90 °C to room temperature, allowing each short staple to find its unique position on the scaffold strand and to ultimately form the desired structure. Voilà!

3D Too!

DNA origami provides support with a well-defined geometry for the 3D lattice of nanoparticles’ growth, as demonstrated in 2016 by Liu et al., who designed a tetrahedron-shaped DNA origami with gold nanoparticles attached to the four vertices as shown here. These nanoparticles then acted as connectors to link the tetrahedron origami-nanoparticle units into a well-ordered face-centered cubic (FCC) lattice structure. If another gold nanoparticle was positioned inside the tetrahedron-shaped origami structure, the final lattice structure achieved would be in the shape of a diamond.

Taken from Hong et al. Chemical Reviews (2017)

LEGO bricks. Taken from

In 2017, Ong et al. published in Nature use of LEGO-like DNA “bricks” with 13-nt binding domains to self-assemble 0.1–1-gigadalton 3D DNA nanostructures from tens of thousands of unique components. To put 1 gigadalton (1 GDa) in perspective, a carbon-12 atom has a mass of 12 Da, a molecule of acetylsalicylic acid (aspirin) has a mass of 180 Da, and Titin—the largest known protein—has a mass of 3,000,000 Da or 3 megadaltons (3 MDa). Giga is a short-form of billion, so 1 GDa is 1 x 109 Da. By my estimate, that’s ~2,000,000 base pairs which predictably self-assemble!

Taken from Ong et al. Nature (2017)

(a) 3D DNA origami are used to construct nanostructures with masses of around 5 MDa from about 200 unique components [circular scaffold (black) and staple (colored) strands]. The DNA brick nanostructures assembled here have masses of up to 500 MDa and contain up to about 30,000 unique components (i.e. bricks). (b) Detailed helical (top) and brick (bottom) models of two 52-nucleotide DNA bricks bound to each other with a 90° dihedral angle via a 13-base-pair interaction. (c) A ~150-MDa DNA brick cuboid (left) consisting of about 10,000 unique components can be used as a molecular “canvas” (middle) with about 20,000 voxels (right), each containing 13 base pairs (see inset). The scale bar for a and c (shown in a) is 100 nm. (d) A 3D rendering of a 0.5 GDa teddy bear (left) can be approximated using the 20,000-voxel canvas (middle) to form the cavity of a cuboid structure (right).

Here’s what Ong et al. stated at the end of their publication:

“Even large DNA brick assemblies might be possible; the high cost of purchasing a large number of synthetic DNA strands restricted our testing to about 30,000 distinct bricks, but low-cost methods for synthesizing DNA strands (such as chip-synthesized DNA followed by parallel enzymatic amplification are available.”

One can only speculate—or dream—about what could be achieved in material science and technology with more massive 3D DNA assemblies constructed from larger DNA bricks. Whatever those achievements might be, I’m pretty sure they’ll be amazing, and won’t necessarily need to be conjured up by sipping a beer like Ned did for his “epiphany”.

As usual, your comments are welcomed.

DNA Day 2018—April 25

  • There are Now Nearly 2 Million DNA-Related Publications 
  • Cheaper Synthesis and Sequencing of DNA Drives Novel Applications 
  • Dreams of Dense Digital Information Storage in DNA Become Reality  

Before getting to the topics that I selected for this post in recognition of DNA Day on April 25, here are some numbers for DNA publications that, to me, are mind-numbing. NIH’s PubMed lists nearly 2 million DNA-related publications, with many more not counted in that database. Looking at the annual contributions to these 2 million articles reveals about 90,000 such publications in just the past year. This computes to nearly 250 per day, 365 days per year, or roughly 10 publications per hour, around the clock!

Taken from

Rationale for Information Storage in DNA

Long-term storage and access of information for use by future generations—think Mr. Spock’s library computer workstation in Star Trek—presents a serious challenge, regardless of data format. This challenge was noted by Bancroft et al. in 2001 in venerable Science magazine as follows:

“[Digital] data currently being stored in magnetic or optical media will probably become unrecoverable within a century or less, through the combined effects of hardware and software obsolescence and decay of the storage medium. New approaches are required that will permit retrieval of information stored for centuries or even millennia.”

These authors proposed that DNA has three properties that lend to its serving as a vehicle for long-term information storage:

  • Storage of DNA can result in extremely long stability, as evidenced by the reported recovery of viable bacteria from 250-million-year-old salt crystals.
  • Because DNA is our genetic material, methods for both storage and reading of DNA-encoded information should remain central to technological civilizations and undergo continual improvements.
  • Use of DNA as a storage can use an enormous number of identical molecules, thus providing extensive informational redundancy to strongly mitigate effects of any losses due to chemical degradation.

Taken from

DNA is an incredibly dense storage medium, potentially squeezing in a mind-boggling 5.5 x 1015 bits (petabits, Pb) or 125,000 x 109 bytes (gigabytes, GB; 1 byte = 8 bits) of information per cubic millimeter. By that measure, it has been estimated that all 700 x 1018 bytes of today’s accessible internet would fit into a space the size of a shoebox!

Demonstrations of Information Storage in DNA

According to a report in Science in 2012 by a team including uber-famous George Church, storing messages in DNA was first demonstrated in 1988, and the largest project following that achieved encoding only 7920 bits in 2010. In comparison, Church and coworkers introduced new technology to demonstrate storage of ~5,300,000 bits of information contained in a book that included 53,426 words, 11 JPG images and 1 JavaScript program. This information was encoded onto ~55,000 159-nt oligos synthesized by ink-jet printing on DNA microchips, which I’ll comment on below. To read the encoded book, PCR was followed by high-throughput sequencing. Interested readers can consult this report for details and a discussion of why this approach offers numerous advantages over previous strategies.

Shortly thereafter in 2013, a team at Wellcome Trust Genome Campus working with Agilent Technologies received widespread media attention for a report on encoding in DNA, and then reading, the entire set of Shakespearean sonnets, a 26-second clip of Martin Luther King’s famous “I have a dream” speech, and a photograph using an approach depicted here.

Fast-forward to March 2018 and we are brought to a publication by Organick et al. in Nature Biotechnology that received loads of media coverage because of its landmark achievement demonstrating random access of information in large-scale DNA data storage. This team from various institutions, including Microsoft, noted that recovering stored data on a large-scale currently requires all the DNA in a pool to be sequenced, even if only a subset of the information needs to be extracted. In contrast, they demonstrated a methodology to encode and store 35 distinct files [over 200 x 106 bytes (megabytes; MB) of data]—including video, audio, images, and text—in more than 13 million DNA oligos. Moreover, they showed recovery of each file individually with no errors, using a random-access approach based on sequence tags for files for specific PCR amplification. Interested readers can consult the paper for details.

Future Prospects

Organick et al. conclude that synthetic DNA production efficiency will have to significantly increase if DNA is to become a practical medium for data storage. They contend that this will be attainable because the synthetic DNA needed for data storage can be much more error prone than DNA required by life sciences, and very few copies per sequence are required (i.e. orders of magnitude less DNA than conventional nanomole scale solid-phase synthesis). This is due to error-correcting algorithms such as the one they described in their paper.

While ink-jet printed DNA oligos using Caruther’s phosphoramidite chemistry can be scaled and improved, template-independent enzymatic oligonucleotide synthesis (TiEOS) is receiving attention because of potential cost reductions. For example, Michael Jensen and uber-famous Ron Davis at Stanford, in a very recently published review, have estimated costs for phosphoramidite vs. TiEOS methods. They concluded that cyclical two-step (couple, deblock) solid-phase femtomole-scale synthesis of DNA oligos by TiEOS with dNTPs (X = H) with 3’-protecting (Pr) groups and terminal deoxynucleotide transferase (TdT) could provide oligos to users at a lower cost by orders of magnitude per nucleotide incorporated.

Taken from Jansen & Davis Biochemistry (2018)

Regarding sequencing of DNA to retrieve stored information, Organick et al. used conventional Illumina reversible terminators as well as newer Oxford Nanopore Technologies’ nanopore sequencing. They noted that “[t]he compactness and potential for scalability makes nanopore-based sequencing an intriguing option for integration in future stand-alone DNA data storage systems”.

I’m optimistic about the future prospects for using DNA to store information. Whether or not you share that optimism, I hope you’ll agree with my opinion that exploratory research toward that goal is yet another example of DNA’s wondrous properties inspiring quests for new technologies.

As usual, your comments are welcomed.

DNA Day Archive

Here are the topics and links to all of my previous blogs on DNA Day for you to reread or check out. Enjoy!

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!

2017—Some of the Top 5 Cited Papers on DNA Will Surprise You


Reproduction of the following announcement in this TriLink BioTechnologies blog was requested of Jerry Zon by Yogesh Sanghvi on behalf of the International Society of Nucleosides, Nucleotides & Nucleic Acids (IS3NA). The stated aim of the IS3NA is to capitalize on the knowledge of practicing members across several disciplines to understand the impact of nucleic acids in a plethora of cutting edge scientific questions ranging from the origins of life to the development of novel therapeutics. Importantly, in my opinion, the IS3NA also aims to act as a mediator for communication, cooperation and understanding between scientists of all nationalities, and to provide information about and to stimulate interest in the above-mentioned areas among persons of all nationalities.


Kool’s Cool Chemistry

  • Eric T. Kool Has a Long-String of Widely Cited Papers on Innovative Nucleic Acid Chemistry   
  • Kool’s Latest Contribution Involves Reversible Chemistry for PhotoCloaking RNA
  • Kool’s Application of This to Aptamers is Very Cool, In My Opinion

Eric T. Kool (taken from

Readers of my blog know that my writing style tends toward using alliterations, which is evident in this blog’s title referring to Prof. Eric T. Kool and his chemistry, and that cool is the slang sense, i.e. awesome, swell, nifty, etc.

At the outset, I should say that Stanford University-based Kool has a long string of very ingenious publications in diverse aspects of nucleic acids, which attract many readers evidenced by ~1,400 citations per year according to Google Scholar metrics. Kool has received numerous awards and honors that are listed at The Kool Lab website, together with his lab’s research interests.

An appreciation of the diversity of Kool’s scientific contributions can be gleaned by later perusing his ~270 publications at this PubMed link, but for now I’ll be focusing on his most recent publication titled RNA Control by Photoreversible Acylation. This article requires—unfortunately—a paid subscription to J. Amer. Chem. Soc. or purchasing access to a pdf, so I’ve tried my best to give you herein a synopsis of what Kool has achieved in this study, and why it’s so cool.

Caged Nucleic Acids

One of the ways to externally initiate (i.e. turn on) nucleic acid biochemical or biological function in vitro or in vivo is to use a “caging” strategy. The term caging generally refers to installation of one or more removable groups on a molecule in such manner so as to render the molecule inactive. For nucleic acids (and proteins, etc.) this has typically been achieved with photoremovable groups, such that irradiation with UV light removes the caging group and restores functional activity.

Taken from Deiters and coworkers ChemBioChem 2008

An illustrative example of this, published by Deiters and coworkers in 2008, is shown here for controlling gene silencing in mammalian cells with light-activated antisense agents. In this case, phosphorothioate-modified antisense agents were rendered inactive by installation of NPOM groups on nucleobases to prevent hybridization to a target mRNA. Brief irradiation with light at 365 nm removes the NPOM caging groups, enables sequence-specific binding to mRNA, and thus, blocks translation and/or leads to RNase H-catalyzed mRNA degradation.

A similar approach has been applied to functional studies of RNA. However, obtaining such photocaged DNA or RNA has usually required synthesis of chemically modified phosphoramidite monomer building blocks for solid-phase synthesis of the desired oligonucleotides. This limits utility to investigators who are able to deal with organic synthesis or have budgets to purchase relative costly photocaged monomers or custom synthesized photocaged oligonucleotides. Applying photocaging to long RNA is even more problematic.

Kool’s Cool Chemistry

To address these obstacles, Kool set out to develop a general method that can be applied post-synthetically to synthetic and native RNAs regardless of length, and moreover could be easily carried out by non-chemists. Based on studies (cited earlier) by others showing that 2’-OH groups of RNA can be selectively acylated in aqueous buffers with activated acyl compounds, Kool envisaged a strategy depicted here. An appropriately designed “PhotoCloaking” agent (PCA) is reacted with 2’-OH groups post-synthetically to block structure and interactions, but are rendered photo-responsive by including photocleavable bonds.

In his publication, Kool reasoned that “addition of several such blocking groups to the RNA (‘cloaking’) would in principle cover and protect it from folding and interacting with other molecules. Subsequently, the acylation could be reversed by exposing RNA to light, removing the blocking groups and switching on activity.” The PCA design strategy included combination of a reactive acyl group with an o-nitroveratryl photo-labile group, as shown here for PCAs 1 and 2 having unmethylated and methylated veratryl groups, respectively, to control the efficiency of photocleavage. Also included was a dimethylaminoethyl group to enhance solubility, and (after testing) 2-chloroimidazole as having the ideal level of reactivity.

A short 12-mer synthetic RNA oligo was used to evaluate 2’-OH acylation in water under mild conditions, in conjunction with denaturing polyacrylamide gel electrophoresis (PAGE) and mass spectrometry (MS), which indicated polyacylation with up to five groups (aka labels). Next, uncloaking of RNA by photoremoval of the PCA labels in water by irradiation with 365 nm light was confirmed by PAGE and MS. A 16-mer synthetic RNA oligo was similarly polyacylated and shown by native gel analysis and UV to have diminished ability to hybridize to complementary DNA. The reactivity of a synthetic hammerhead ribozyme previously reported was similarly disabled by PhotoCloaking and subsequently restored by exposure to UV light.

Kool then hypothesized that this postsynthetic polyacylation strategy would offer a convenient and simple method for preparing a photocontrolled aptamer of any length, which I have touted as a useful class of compounds in previous blogs. Kool’s test for this was especially interesting, in my opinion, because he used a 150-nt RNA transcript called Broccoli. This oddly named RNA is an aptamer that folds into a compact tertiary structure which binds DFHBI dye giving rise to a strong increase in fluorescence, and thus serves as an RNA mimic of well-known green fluorescent protein.

Kool had to use the more reactive PCA 2 to prepare a suitably cloaked version of Broccoli, which exhibited very dim fluorescence compared to uncloaked (i.e. untreated) Broccoli. When the cloaked aptamer was exposed to UV light and incubated with DFHBI, fluorescence was completely restored to the level of the untreated sample. The capability to use this PhotoCloaking strategy to switch on RNA function in cells was assessed by transfecting a 237-nt cloaked Broccoli-dimer RNA construct into a human (HeLa) cell line and then exposing these cells to light. As can be seen from the results shown here, the cloaking and uncloaking worked just as intended.

Epifluorescence microscopy of HeLa cells transfected with untreated or cloaked 237-nt Broccoli. Taken from Kool and coworkers J Am Chem Soc 2018

Kool concluded that “a great advantage of this postsynthetic labeling is that it allows for one-step photoprotection of long, biologically relevant RNAs that are difficult or impossible to synthesize via solid-state oligonucleotide synthesis. Since PhotoCloaking can be achieved without specialized equipment, the new method could potentially find widespread use for study of the biology of RNAs.”

Very cool indeed!

As usual, your comments are welcomed.