Multiplexed Programmable Release of Captured DNA

Kennedy‐Darling, Julia; Holden, Matthew T.; Shortreed, Michael R.; Smith, Lloyd M.

doi:10.1002/cbic.201402343

Cited by 8 publications

(6 citation statements)

References 24 publications

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“…The capture oligonucleotide hybridizes to the target RNA-protein complexes and the hybrid is isolated from the lysate using streptavidin-coated magnetic beads. Following stringent washes the RNA-protein complexes are released from the beads using a toehold release oligonucleotide 34 . This oligonucleotide is complementary to all 38 nucleotides of the capture oligonucleotide and is therefore thermodynamically favored to hybridize with it over the target RNA (which is only complementary to 30 nucleotides of the capture oligonucleotide), thereby releasing the HIV RNA-protein complex into solution (Fig.…”

Section: Resultsmentioning

confidence: 99%

Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry

Knoener

Becker

Scalf

et al. 2017

Sci Rep

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HIV-1 replication requires myriad interactions between cellular proteins and the viral unspliced RNA. These interactions are important in archetypal RNA processes such as transcription and translation as well as for more specialized functions including alternative splicing and packaging of unspliced genomic RNA into virions. We present here a hybridization capture strategy for purification of unspliced full-length HIV RNA-protein complexes preserved in vivo by formaldehyde crosslinking, and coupled with mass spectrometry to identify HIV RNA-protein interactors in HIV-1 infected cells. One hundred eighty-nine proteins were identified to interact with unspliced HIV RNA including Rev and Gag/Gag-Pol, 24 host proteins previously shown to bind segments of HIV RNA, and over 90 proteins previously shown to impact HIV replication. Further analysis using siRNA knockdown techniques against several of these proteins revealed significant changes to HIV expression. These results demonstrate the utility of the approach for the discovery of host proteins involved in HIV replication. Additionally, because this strategy only requires availability of 30 nucleotides of the HIV-RNA for hybridization with a capture oligonucleotide, it is readily applicable to any HIV system of interest regardless of cell type, HIV-1 virus strain, or experimental perturbation.

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Section: Resultsmentioning

confidence: 99%

Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry

Knoener

Becker

Scalf

et al. 2017

Sci Rep

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“…The beads were then concentrated into a smaller volume and washed once for 5 min and once for 1 h. To release the hybridized complexes, a toehold-mediated strand displacement strategy was employed (see Supplementary Information for details). This strategy was developed and demonstrated for the selective release of individual target loci from cross-linked yeast chromatin, 27,28 and here we extended the strategy to cross-linked human chromatin. First, alpha satellite release solution containing equimolar concentrations of the eight alpha satellite release oligonucleotides was added to the beads, and the resultant bead slurry was gently mixed for 15 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Elucidating Protein–DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry

Buxton¹,

Kennedy‐Darling²,

Shortreed³

et al. 2017

J. Proteome Res.

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The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.

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“…These probe–lncRNA-RBP complexes are isolated with streptavidin-coated magnetic beads [ 77 ]. After the simultaneous capture of multiple targets, each target is released in a sequence-specific manner, referred to as the toehold-mediated release [ 94 , 95 ]. Each probe harbors an additional 8-nt sequence, which does not hybridize the target, called the toehold sequence.…”

Section: Overview Of Available Rna- and Rnp-centric Purification Mmentioning

confidence: 99%

Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

2020

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The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).

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Multiplexed Programmable Release of Captured DNA

Cited by 8 publications

References 24 publications

Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry

Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry

Elucidating Protein–DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry

Single and Combined Methods to Specifically or Bulk-Purify RNA–Protein Complexes

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