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Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The R17 complex I was studied exhaustively by Olke C. Uhlenbeck's team in Urbana, Illinois, and further in Boulder, Colorado. They were the first to conduct the entire enzymatic synthesis of 21-nucleotide fragment corresponding to the positions from −17 to +4 of the replicase gene (Krug et al. 1982). By the synthesis, the T4 RNA ligase was used to join shorter oligomers. This sequence was identical with the R17 replicase initiator region and also encompassed the binding domain of the R17 coat protein. The resulting fragment had a secondary structure with the expected thermal stability, and demonstrated the same affinity by the coat binding as the 59-nucleotide fragment isolated from the R17 RNA and described above (Krug et al. 1982). The kinetic and equilibrium properties of the interaction between the 21-nucleotide RNA operator and the R17 coat were studied by the filter retention assay (Carey and Uhlenbeck 1983). The kinetics of the reaction were consistent with the equilibrium association constant and indicated a diffusion-controlled reaction. The temperature dependence of Ka gave ΔH = −19 kcal/mol. This large favorable ΔH was partially offset by a ΔS = −30 cal mol−1 deg−1 to give a ΔG = −11 kcal/mol at 2°C in 0.19 M salt. The binding reaction had a pH optimum centered around pH 8.5, but pH had no effect on the ΔH. While the interaction was insensitive to the type of monovalent cation, the affinity decreased with the lyotropic series among monovalent anions. The ionic strength dependence of Ka revealed that ionic contacts contributed to the interaction. Most of the binding free energy, however, was regarded as a result of nonelectrostatic interactions (Carey and Uhlenbeck 1983).
Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD)
Published in Journal of Extracellular Vesicles, 2020
Goren Saenz-Pipaon, Patxi San Martín, Núria Planell, Alberto Maillo, Susana Ravassa, Amaia Vilas-Zornoza, Esther Martinez-Aguilar, José Antonio Rodriguez, Daniel Alameda, David Lara-Astiaso, Felipe Prosper, José Antonio Paramo, Josune Orbe, David Gomez-Cabrero, Carmen Roncal
RNA-Seq was performed in EVs (details are provided in Supplemental Methods) from controls, PAD patients with intermittent claudication (IC, Fontaine class IIa) and PAD patients with critical limb ischaemia (CLI, Fontaine class IV) with myocardial infarction in the follow-up study (n = 12/group). The protocol was adapted from Jaitin et al., 2014 (MARS-Seq) [16]. Briefly, 50 µL of isolated EVs were mixed with 50 µL of Lysis/Binding Buffer (Invitrogen). Poly-A RNA was captured with Dynabeads Oligo (dT) (Invitrogen) and reverse-transcribed with AffinityScript Multiple Temperature Reverse Transcriptase (Agilent) using oligo (dT) primers carrying a 7 bp index. Up to eight samples with similar overall RNA content were pooled together and subjected to linear amplification by in vitro transcription using a HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). Amplified RNA was fragmented into 250–350 bp with RNA Fragmentation Reagents (Invitrogen) and dephosphorylated with thermosensitive alkaline phosphatase (FastAP, Thermo). Partial Illumina adaptor sequences [16] were ligated with T4 RNA Ligase 1 (New England Biolabs), followed by a second reverse transcription reaction. Full Illumina adaptor sequences were added with KAPA HiFi DNA Polymerase (Kapa Biosystems). Libraries were sequenced in an Illumina NextSeq 500 at a sequence depth of 10 million reads per sample. All RNA-Seq data have been submitted to NCBI GEO repository, study number GSE140320.
The expression of circulating hsa-miR-126-3p in dengue-infected Thai pediatric patients
Published in Pathogens and Global Health, 2023
Methee Sriprapun, Jittraporn Rattanamahaphoom, Pimolpachr Sriburin, Supawat Chatchen, Kriengsak Limkittikul, Chukiat Sirivichayakul
First-strand (cDNA) synthesis was performed using TaqMan™ Advanced miRNA cDNA Synthesis Kit (Applied Biosystems: ThermoFisher Scientific, Massachusetts, USA) by following the instruction manual and all processes were run with PCR thermocycler (GeneAmp™ PCR System 9700, Applied Biosystems: ThermoFisher Scientific, Massachusetts, USA). The reaction is composed of poly-A tail addition, adaptor ligation reaction, reverse transcription, and pre-amplification. Briefly, poly-A tail synthesis was performed by adding 2 μL of mastermix composing of 10X poly (A) buffer, ATP, poly (A) enzyme into 3 µL of extracted miRNA. Polyadenylation reaction was performed at 37°C for 45 minutes, then the reaction was terminated at 65°C for 10 minutes. Adapter ligation was continued by adding 5X DNA ligase buffer, 50% PEG 8000, 25X ligation adapter, and RNA ligase. The ligation reaction was set at 16°C for 60 minutes. The reverse transcription step was done after finishing the ligation step by using 5X reverse transcription (RT) buffer, 25 mM dNTP mix, 20X universal RT primer, and 10X reverse transcription enzyme mix. The reaction was incubated at 42°C for 15 minutes and enzymatic activity was terminated at 85°C for 5 minutes. In order to increase the sensitivity of miRNA detection, miR-Amp reaction was performed by adding 5 μL of cDNA with 45 μL of PCR reagent containing 2X miR-Amp mastermix and 20X miR-Amp primer mix. Fourteen cycles of PCR reaction were done in the condition of 95°C for 5 minutes (enzyme activation), 95°C for 3 seconds (denaturation), 60°C for 30 seconds (annealing/extension), and 99°C for 10 minutes for terminating the reaction. The PCR product was kept at −20°C up to 2 months prior to performing quantitative PCR.
Recombinant bacteriophage T4 Rnl1 impacts Streptococcus mutans biofilm formation
Published in Journal of Oral Microbiology, 2021
Juxiu Chen, Zhanyi Chen, Keyong Yuan, Zhengwei Huang, Mengying Mao
The ecology of the oral microbiome significantly affects the oral health status. Beyond bacteria, bacteriophages are thought to play a critical role in shaping the oral microbiome [1]. Recently, the increasing attention has been given to the phage therapy. Phages can be used to attack the target pathogens and prevent oral infectious disease development, such as the most prevalent, dental caries [2]. It has been reported that a new phage, ɸAPCM01, can infect Streptococcus mutans DPC6143, one of the principal agents of dental caries formation, further impacting on its growth and biofilm formation [3]. Bacteriophages are highly evolved nanomachines that recognize bacterial cell walls to deliver genetic information [4]. However, the exquisite specificity against pathogens limits phage therapy to common use [5]. Bacteriophage T4 RNA ligase 1 (T4 Rnl1) is a tRNA repair enzyme that functions in phage infection and has been found to be stably expressed and work in many bacteria via recombinant vector [6–8]. The expression of bacteriophage virulent proteins like T4 Rnl1 in target bacteria by a recombinant vector might provide an alternative strategy to overcome the narrow spectrum of the traditional phage therapy. As the founding member of the RNA ligase family, T4 Rnl1 can facilitate a preferential ligation of the 3ʹ and 5ʹ ends of RNA and has been used as a biological tool used for identifying direct targets of bacterial small regulatory RNA in many bacteria [9,10]. The expressed recombinant T4 Rnl1 could negatively affect cell viability in Pseudomonas aeruginosa [6,8]. Recently, we firstly established the T4 Rnl1 expression system in S. mutans for detecting the targets of sRNAs [7]. However, the biofunctional effect of recombinant T4 Rnl1 on S. mutans remains unclear.