rna analysis

The Molecular Blueprint for Virus-Resistant Cowpea

Posted on by Anna Bennett

Cowpea (Vigna unguiculata), a humble tan and black legume, is one of the most important food crops in the world. Grown across sub-Saharan Africa, Asia, and parts of the Americas, Cowpea provides protein-rich nutrition for hundreds of millions of people, making it a cornerstone of smallholder agriculture. But cowpea production faces a persistent threat: the cowpea aphid-borne mosaic virus (CABMV), a common virus that can devastate yields across entire growing regions.

A dark background with a wooden spoon holding tan and black beans scattered on the spoon and on the background.

What makes CABMV particularly difficult to combat is how the virus infects its host. Instead of relying on viral translational machinery, the virus hijacks the plant’s systems to replicate. CABMV targets a protein called eIF4E, a translation initiation factor that the plant needs to read its own genetic instructions and produce proteins. The virus produces a protein, VPg, that binds directly to eIF4E and redirects the plant’s translational machinery to produce viral proteins instead. The plant can’t simply get rid of eIF4E. Without it, protein synthesis stalls. So how can cowpea defend itself against a virus that exploits one of its most essential proteins?

A new study published in Agronomy by researchers at the Federal University of Pernambuco, the Federal University of Minas Gerais, and Embrapa Recursos Genéticos e Biotecnologia takes a comprehensive look at this problem from the inside out1. The team characterized all three members of the eIF4E gene family in cowpea  (eIF4E, eIF(iso)4E, and nCBP) across six cultivated varieties (cultivars) with known contrasting responses to CABMV infection. Two of those cultivars (Bajão and IT85F-2687) are resistant to the virus; the other four (Boca Negra, BR14 Mulato, Pingo de Ouro, and Santo Inácio) are susceptible to the virus.

Using a multi-omics approach that combined genomic, evolutionary and structural analyses, the researchers set out to answer a fundamental question: what makes some versions of eIF4E exploitable by the virus, and others not?

Continue reading “The Molecular Blueprint for Virus-Resistant Cowpea”

Sequence to Substance: Making the mRNA Therapeutic

Posted on by Shannon Sindermann

mRNA-based therapeutics are being explored across a range of applications, including vaccines, protein replacement and immunotherapies (2).

Before any formulation decisions enter the picture, teams need confidence in the RNA itself: that it is the right sequence, right properties and the right purity to behave predictably downstream. That is where it helps to separate drug substance from drug product. The drug substance is the active ingredient intended to deliver a pharmacological effect, while drug product is the finished dosage form that contains that ingredient (6).

This post focuses on what happens upstream, making the mRNA drug substance before formulation. In practical terms, that upstream work spans choosing an mRNA construct, producing it by IVT, and then purifying and analyzing the product so it has the desired quality attributes (5).

Continue reading “Sequence to Substance: Making the mRNA Therapeutic”

Ancient RNA From a Woolly Mammoth?

Posted on by Shannon Sindermann

Most of us first meet woolly mammoths as Manny from Ice Age (a gentle giant with main character energy) or as towering skeletons in museum halls. In the lab, though, mammoths can show up in many ways: such as fragile molecules preserved in permafrost for tens of thousands of years.

Woolly Mammoth

Ancient DNA has already helped scientists piece together mammoth genomes. Now scientists have done something wilder: they’ve pulled ancient RNA out of a ~39,000-year-old woolly mammoth and used it to see which genes were being expressed in its muscle tissue. In a new study, researchers showed that not only can woolly mammoth DNA survive tens of thousands of years in permafrost, but RNA, the fragile, quick-to-degrade “live feed” of the cell, can too.

Continue reading “Ancient RNA From a Woolly Mammoth?”

Riboprobes: RNA Probes Are Still Valuable Research Tools

Posted on by Gary Kobs

9613ca[1]Riboprobes are RNA probes that can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
Viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.
Transcription of RNA is performed with the appropriate RNA polymerase (T3, T7 or SP6), depending on the RNA polymerase promoter sites present in the chosen vector. Because these polymerases are extremely promoter-specific (i.e., there is almost no transcriptional cross talk), virtually homogeneous RNA can be obtained using plasmid DNA as the template in a transcription reaction. When it is desirable to copy only insert DNA sequences, the plasmid is linearized at an appropriate restriction site before the transcription reaction and only discrete “run-off” transcripts are obtained, virtually free of vector sequences. RNA transcripts may be used to generate radioactive probes for hybridization to Northern and Southern blots, plaque and colony lifts as well as non-radioactive probes (i.e, labeled with digoxgenin)for in situ hybridization.

Recent references using riboprobes include: Continue reading “Riboprobes: RNA Probes Are Still Valuable Research Tools”