FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes
The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or t...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society
2013
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| Online Access: | http://hdl.handle.net/1721.1/79407 https://orcid.org/0000-0002-2100-1171 https://orcid.org/0000-0001-5629-4798 https://orcid.org/0000-0003-4255-0492 |
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| author | Alabi, Christopher A. Love, Kevin T. Sahay, Gaurav Stutzman, Tina M. Young, Whitney T. Langer, Robert Anderson, Daniel Griffith |
| author2 | Harvard University--MIT Division of Health Sciences and Technology |
| author_facet | Harvard University--MIT Division of Health Sciences and Technology Alabi, Christopher A. Love, Kevin T. Sahay, Gaurav Stutzman, Tina M. Young, Whitney T. Langer, Robert Anderson, Daniel Griffith |
| author_sort | Alabi, Christopher A. |
| collection | MIT |
| description | The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (KD) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing. |
| first_indexed | 2024-09-23T08:12:47Z |
| format | Article |
| id | mit-1721.1/79407 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:12:47Z |
| publishDate | 2013 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/794072022-09-23T11:37:39Z FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes Alabi, Christopher A. Love, Kevin T. Sahay, Gaurav Stutzman, Tina M. Young, Whitney T. Langer, Robert Anderson, Daniel Griffith Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Chemical Engineering Koch Institute for Integrative Cancer Research at MIT Alabi, Christopher Akinleye Alabi, Christopher A. Love, Kevin T. Sahay, Gaurav Stutzman, Tina M. Langer, Robert Anderson, Daniel Griffith The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (KD) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing. National Institutes of Health (U.S.) (NIH grant R37-EB000244) National Institutes of Health (U.S.) (NIH grant R01-CA132091) National Institutes of Health (U.S.) (NIH grant R01-CA115527) National Institutes of Health (U.S.) (Postdoctoral Fellowship) 2013-07-02T16:15:16Z 2013-07-02T16:15:16Z 2012-06 2012-03 Article http://purl.org/eprint/type/JournalArticle 1936-0851 1936-086X http://hdl.handle.net/1721.1/79407 Alabi, Christopher A., Kevin T. Love, Gaurav Sahay, Tina Stutzman, Whitney T. Young, Robert Langer, and Daniel G. Anderson. FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA Nanocomplexes. ACS Nano 6, no. 7 (July 24, 2012): 6133-6141. https://orcid.org/0000-0002-2100-1171 https://orcid.org/0000-0001-5629-4798 https://orcid.org/0000-0003-4255-0492 en_US http://dx.doi.org/10.1021/nn3013838 ACS Nano Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society Christopher Alabi |
| spellingShingle | Alabi, Christopher A. Love, Kevin T. Sahay, Gaurav Stutzman, Tina M. Young, Whitney T. Langer, Robert Anderson, Daniel Griffith FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes |
| title | FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes |
| title_full | FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes |
| title_fullStr | FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes |
| title_full_unstemmed | FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes |
| title_short | FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes |
| title_sort | fret labeled sirna probes for tracking assembly and disassembly of sirna nanocomplexes |
| url | http://hdl.handle.net/1721.1/79407 https://orcid.org/0000-0002-2100-1171 https://orcid.org/0000-0001-5629-4798 https://orcid.org/0000-0003-4255-0492 |
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