More Like this

1. Non‐Chromatographic Purification of Synthetic RNA Using Bio‐Orthogonal Chemistry

   https://doi.org/10.1002/cpz1.247  

   He, Muhan ; Wu, Xunshen ; Mao, Song ; Haruehanroengra, Phensinee ; Khan, Irfan ; Sheng, Jia ; Royzen, Maksim ( September 2021 , Current Protocols)

   Solid‐phase synthesis of RNA oligonucleotides over 100 nt in length remains challenging due to the complexity of purification of the target strands from the failure sequences. This article describes a non‐chromatographic procedure that will enable routine solid‐phase synthesis and purification of long RNA strands. The optimized five‐step process is based on bio‐orthogonal inverse electron demand Diels‐Alder chemistry betweentrans‐cyclooctene (TCO) and tetrazine (Tz), and entails solid‐phase synthesis of RNA on a photo‐labile support. The target oligonucleotide strands are selectively tagged with Tz while on‐support. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach can be applied for purification of 76‐nt long tRNA and 101‐nt long sgRNA for CRISPR experiments. Purity of the isolated oligonucleotides should be evaluated using gel electrophoresis, while functional fidelity of the sgRNA should be confirmed using CRISPR‐Cas9 experiments. © 2021 Wiley Periodicals LLC.

   Basic Protocol: Five‐step non‐chromatographic purification of synthetic RNA oligonucleotides

   Support Protocol 1: Synthesis of the components that are required for the non‐chromatographic purification of long RNA oligonucleotides.

   Support Protocol 2: Solid‐phase RNA synthesis

    

   more » « less
2. Temporal and spatial transcriptomic and micro RNA dynamics of CAM photosynthesis in pineapple

   https://doi.org/10.1111/tpj.13630  

   Wai, Ching M. ; VanBuren, Robert ; Zhang, Jisen ; Huang, Lixian ; Miao, Wenjing ; Edger, Patrick P. ; Yim, Won C. ; Priest, Henry D. ; Meyers, Blake C. ; Mockler, Todd ; et al ( August 2017 , The Plant Journal)

   The altered carbon assimilation pathway of crassulacean acid metabolism (CAM) photosynthesis results in an up to 80% higher water‐use efficiency than C₃photosynthesis in plants making it a potentially useful pathway for engineering crop plants with improved drought tolerance. Here we surveyed detailed temporal (diel time course) and spatial (across a leaf gradient) gene and microRNA(miRNA) expression patterns in the obligateCAMplant pineapple [Ananas comosus(L.) Merr.]. The high‐resolution transcriptome atlas allowed us to distinguish betweenCAM‐related and non‐CAMgene copies. A differential gene co‐expression network across green and white leaf diel datasets identified genes with circadian oscillation,CAM‐related functions, and source‐sink relations. Gene co‐expression clusters containingCAMpathway genes are enriched with clock‐associatedcis‐elements, suggesting circadian regulation ofCAM. About 20% of pineapple microRNAs have diel expression patterns, with several that target keyCAM‐related genes. Expression and physiology data provide a model forCAM‐specific carbohydrate flux and long‐distance hexose transport. Together these resources provide a list of candidate genes for targeted engineering ofCAMinto C₃photosynthesis crop species.

    

   more » « less
3. Template‐Directed Copying of RNA by Non‐enzymatic Ligation

   https://doi.org/10.1002/ange.202004934  

   Zhou, Lijun ; O'Flaherty, Derek K. ; Szostak, Jack W. ( August 2020 , Angewandte Chemie)

   The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.

    

   more » « less
4. Template‐Directed Copying of RNA by Non‐enzymatic Ligation

   https://doi.org/10.1002/anie.202004934  

   Zhou, Lijun ; O'Flaherty, Derek K. ; Szostak, Jack W. ( August 2020 , Angewandte Chemie International Edition)

   The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.

    

   more » « less
5. Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion

   https://doi.org/10.1186/s12864-020-07134-4  

   Wangsanuwat, Chatarin ; Heom, Kellie A. ; Liu, Estella ; O’Malley, Michelle A. ; Dey, Siddharth S. ( October 2020 , BMC Genomics)

   RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification.

   EMBR-seq results in 90% of the sequenced RNA molecules from anE. coliculture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA.

   EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.

    

   more » « less