TL Spotlight on TriLink Product Applications

• Nearly 500 Publications in 2017 Cite Use of TriLink Products
• Jerry Spotlights 20 Citing Oligos, Nucleotides, mRNA and Aptamers
• 10 of These 20 Spotlighted Items Show Global Reach of TriLink Products

While thinking about possible topics to blog about, it occurred to me that researching recent publications on the applications of TriLink products would likely lead to many options. Using Google Scholar to do just that, I was given nearly 500 items, which is indeed plenty. However, choosing which to feature was neither an easy nor objective task. Having said that, and with sincere apologies to publications not spotlighted here, my “faves” and comments are given below, listed arbitrarily (not ranked) in four product categories: oligonucleotides, nucleotides, mRNA, and aptamers.

For convenience, each publication title can be clicked on to access the original article. Links to the cited TriLink products are also provided, alongside links to other adjunct information. Several trending “hot topics” and previous blogs are also noted.

Oligonucleotides

• Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors. These researchers at Gilead Sciences, a leading drug company, characterized endonuclease activity using a fluorogenic assay of the internally quenched fluorescent RNA substrate 5’-GAAUA(FAM)UGCAUCAUAGCAUCC(Dabcyl)-3’.
• Fragile X mental retardation protein recognizes a G quadruplex structure within the survival motor neuron domain containing 1 mRNA 5′-UTR. Using primer and template DNA from TriLink, academic collaborators found that the G quadruplex RNA (not DNA) structure present in the 5′-UTR of mRNA influences protein synthesis machinery, as depicted here.

• Enhancement of fluorescent resonant energy transfer and the antenna effect in DNA structures with multiple fluorescent dyes. This research led by Michael J. Heller, who pioneered FRET, is a “must read” for all users of FRET.
• Anti-microRNA targeting using peptide-based nanocomplexes to inhibit differentiation of human pancreatic stellate cells. This study by researchers in The Netherlands used scrambled miRNA and anti-miRNA 21-mer sequences with 2′ O-methyl modifications.
• Ultrafast spectroscopy on DNA-cleavage by endonuclease in molecular crowding. Researchers in India used HPLC-grade oligonucleotides; here’s a link to ~4,000 molecular crowding studies published during the last ~4 years, which represent a trending hot topic.

Nucleotides

• Modification of 3′ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity. Like TdT, DNA pol θ can add modified dNTPs (e.g. biotin-16-Aminoallyl-2'-dUTP) to 3’ termini.
• Directed evolution of protein thermal stability using yeast surface display. An Austrian-led collaboration used 8-Oxo-2´-deoxyguanosine-5´-triphosphate (8-oxo-dGTP) and 2´-deoxy-P-nucleoside-5´-triphosphate (dPTP) modified dNTPs for directed evolution, another trending hot topic.

• Analysis of ribonucleotide 5′-triphosphate analogs as potential inhibitors of Zika virus RNA-dependent RNA polymerase by using nonradioactive polymerase assays. A dozen natural NTPs and dNTPs were used for a screening assay for NTP analogs as candidate inhibitors of Zika virus, which I previously blogged about here.
• Structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs. Researchers at Alios BioPharma used natural nucleoside triphosphates, ATP, CTP, GTP and UTP in this study of mitochondrial toxicity induced by antiviral agents.
• Specific Interactions of Antitumor Metallocenes with Deoxydinucleoside Monophosphates Deoxydinucleoside monophosphates [d(ApT), d(TpA), d(ApG), d(GpA), d(CpG), d(GpC), d(CpT), d(TpC)>

  were synthesized by TriLink for this research done in Switzerland.

mRNA

Modified mRNA for new therapeutic approaches continues to be an amazingly hot area of R&D, which I have previous dubbed “modified mRNA mania” in a previous blog. Interested readers can peruse this link to ~300 items found in my Google Scholar search for TriLink and mRNA publications in 2017.

• Synthesis of Modified mRNA for Myocardial Delivery. Modifications were incorporated by use of 5-methylcytidine-5'-Triphosphate (5-me-CTP).
• In vitro transcription and validation of human pancreatic transcription factors' mRNAs. These researchers in Turkey used (5-me-CTP) and pseudouridine-5'-triphosphate (pseudo-UTP), the latter of which has been featured in several earlier blogs.
• Modified hCFTR mRNA restores normal lung function in a mouse model of cystic fibrosis. Modifications in mRNA were incorporated by use of 2-thiouridine-5'-triphosphate (2-thio-UTP) and 5-me-CTP for this German and Italian collaborative study.

• Co-delivery of nucleoside-modified mRNA and TLR agonists for cancer immunotherapy: Restoring the immunogenicity of immunosilent mRNA. In addition to 5-me-CTP and pseudo-UTP, these researchers in Belgium used unmodified and nucleoside-modified (5meC) mRNA encoding firefly luciferase (fLuc), and the Cy5 labelled mRNA construct (5meC) encoding for eGFP, which are now available as new structural versions of stocked reporter gene mRNAs.
• RNA‐Generated and Gene‐Edited Induced Pluripotent Stem Cells (iPSCs) for Disease Modeling and Therapy. This large collaborative team, which included Germans and Italians, report an optimized protocol for producing iPSCs that uses Cas9 mRNA with substituted nucleotides.

Aptamers

• A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice. Biotin was incorporated into the 5′ ends of aptamers by spiking the aptamer transcriptions with 5′-biotin-spacer9-G (biotin-GMP) at 4-fold excess over the GTP.
• Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence. I have previously blogged about the utility of various SELEX methods. In this study, the starting RNA library was generated by in vitro transcription using natural purines, 2′-fluoro-modified pyrimidines (2’-F-dCTP and 2’-F-dUTP) and a modified T7 RNA polymerase.

• Inhibition of Japanese encephalitis virus (JEV) replication by specific RNA aptamer against JEV methyltransferase. These researchers in the Republic of Korea generated an RNA library by in vitro transcription with 2′-O-methyl pyrimidine nucleotides, natural 2'-hydroxyl purine nucleotides, and a mutant T7 RNA polymerase.
• Development of RNA aptamer that inhibits methyltransferase activity of dengue virus. In an analogous investigation by the aforementioned Koreans, an RNA library was generated by in vitro transcription with 2′-fluoro pyrimidine nucleotides, natural 2′-hydroxyl purine nucleotides, and a mutant T7 RNA polymerase.
• A Conjugate of Two tPA-Binding RNA Aptamers Efficiently Inhibits Fibrinolysis. These investigators in Denmark prepared 2’-fluoro modified RNA aptamers using 2′-fluoro pyrimidine nucleotides. This bivalent aptamer construct was superior to monovalent versions for inhibition of tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals.

Global Reach

A pleasantly surprising aspect of the selected-product search results given above is the worldwide distribution of researchers using TriLink products. This global reach, if you will, is evident from the following countries outside of the USA, which I made point of mentioning:

The Netherlands, India, Austria, Switzerland, Turkey, Germany, Italy, Belgium, Republic of Korea, and Denmark.

All of the publications listed above were selected solely on the type of TriLink product used. Given the relatively small “sample size” of these selected publications, which are only 20-of-500, finding investigators in 10 countries outside of the USA is a compelling testimonial for the TriLink global reach.

World Science Day

Truth be told, when I was searching for a fitting image to visually convey the concept of “global science,” I came across the fact that the United Nations Educational, Scientific, and Cultural Organization (UNESCO) has designated November 10 as World Science Day, with an emphasis on peace and development. The stated intention is to highlight “the important role of science in society and the need to engage the wider public in debates on emerging scientific issues. It also underlines the importance and relevance of science in our daily lives.”

According to UNESCO, “[t>

he theme for 2018 is ‘Science, a Human Right’, in celebration of the 70th anniversary of the Universal Declaration of Human Rights (art. 27), and of the Recommendation on Science and Scientific Researchers. Recalling that everyone has a right to participate in and benefit from science, it will serve to spark a global discussion on ways to improve access to science and to the benefits of science for sustainable development.”

To me, this is a long-term objective which is indeed critical for betterment of future generations.

As usual, your comments are welcomed.

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