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A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.

ABSTRACT: Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.

SUBMITTER: Sawant AA

PROVIDER: S-EPMC4737177 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.

Sawant Anupam A AA Tanpure Arun A AA Mukherjee Progya P PP Athavale Soumitra S Kelkar Ashwin A Galande Sanjeev S Srivatsan Seergazhi G SG

Nucleic acids research 20150917 2

Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzym ...[more]

PMID: 26384420

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Project description:Lesioning and neurophysiological studies have facilitated the elucidation of cortical functions and mechanisms of functional recovery following injury. Clinical translation of such studies is contingent on their employment in non-human primates (NHPs), yet tools for monitoring and modulating cortical physiology are incompatible with conventional lesioning techniques. To address these challenges, we developed a toolbox validated in seven macaques. We introduce the photothrombotic method for inducing focal cortical lesions, a quantitative model for designing experiment-specific lesion profiles and optical coherence tomography angiography (OCTA) for large-scale (~5 cm2) monitoring of vascular dynamics. We integrate these tools with our electrocorticographic array for large-scale monitoring of neural dynamics and testing stimulation-based interventions. Advantageously, this versatile toolbox can be incorporated into established chronic cranial windows. By combining optical and electrophysiological techniques in the NHP cortex, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, integrate physiological and behavioral findings, and develop neurorehabilitative treatments. MOTIVATION The primate neocortex encodes for complex functions and behaviors, the physiologies of which are yet to be fully understood. Such an understanding in both healthy and diseased states can be crucial for the development of effective neurorehabilitative strategies. However, there is a lack of a comprehensive and adaptable set of tools that enables the study of multiple physiological phenomena in healthy and injured brains. Therefore, we developed a toolbox with the capability to induce targeted cortical lesions, monitor dynamics of underlying cortical microvasculature, and record and stimulate neural activity. With this toolbox, we can enhance our understanding of cortical functions, investigate functional recovery mechanisms, test stimulation-based interventions, and integrate physiological and behavioral findings.

Project description:PurposeThe present study aims at developing and evaluating an urea-based prostate specific membrane antigen (PSMA) inhibitor suitable for labeling with 111In for SPECT and intraoperative applications as well as 68Ga and 64Cu for PET imaging.MethodsThe PSMA-based inhibitor-lysine-urea-glutamate-coupled to the spacer Phe-Phe-D-Lys(suberoyl) and functionalized with the enantiomerically pure prochelator (R)-1-(1-carboxy-3-carbotertbutoxypropyl)-4,7-carbotartbutoxymethyl)-1,4,7-triazacyclononane ((R)-NODAGA(tBu)3), to obtain (R)-NODAGA-Phe-Phe-D-Lys(suberoyl)-Lys-urea-Glu (CC34). CC34 was labeled with 111In, 68Ga and 64Cu. The radioconjugates were further evaluated in vitro and in vivo in LNCaP xenografts by biodistribution and PET studies. Biodistribution studies were also performed with 68Ga-HBED-CC-PSMA (HBED-CC: N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid) and 111In-PSMA-617 for comparison.Results68Ga-CC34, 64Cu-CC34, and 111In-CC34 were prepared in radiochemical purity > 95%. 68/natGa-CC34, 64/natCu-CC34, 111/natIn-CC34, 68/natGa-HBED-CC-PSMA, and 111/natIn-PSMA-617 exhibited high affinity for the LNCaP cells, with Kd values of 19.3 ± 2.5 nM, 27.5 ± 2.7 nM, 5.5 ± 0.9 nM, 2.9 ± 0.6 nM and 5.4 ± 0.8 nM, respectively. They revealed comparable internalization profiles with approximately 75% of the total cell associated activity internalized after 3 h of incubation. 68Ga-CC34 showed very high stability after its administration in mice. Tumor uptake of 68Ga-CC34 (14.5 ± 2.9% IA/g) in LNCaP xenografts at 1 h p.i. was comparable to 68Ga-HBED-CC-PSMA (15.8 ± 1.4% IA/g) (P = 0.67). The tumor-to-normal tissue ratios at 1 and 2 h p.i of 68Ga-CC34 were also comparable to 68Ga-HBED-CC-PSMA (P > 0.05). Tumor uptake of 111In-CC34 (28.5 ± 2.6% IA/g) at 1 h p.i. was lower than 111In-PSMA-617 (52.1 ± 6.5% IA/g) (P = 0.02). The acquisition of PET-images with 64Cu-CC34 at later time points showed wash-out from the kidneys, while tumor uptake still remained relatively high. This resulted in an increased tumor-to-kidney ratio over time.Conclusions68Ga-CC34 is comparable to 68Ga-HBED-CC-PSMA in terms of tumor uptake and tumor to normal tissue ratios. 64Cu-CC34 could enable high contrast imaging of PSMA positive tissues characterized by elevated expression of PSMA or when delayed imaging is required. 64Cu-CC34 is currently being prepared for clinical translation.

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A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.

Publications

A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA.

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