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Digital imprinting of RNA recognition and processing on a self-assembled nucleic acid matrix.

Redhu SK, Castronovo M, Nicholson AW - Sci Rep (2013)

Bottom Line: The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment.The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input.These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

View Article: PubMed Central - PubMed

Affiliation: Nicholson and MONALISA (MOlecularNAnotechnology for LIfe Science Applications) Laboratories of the Department of Biology, Temple University, 1901 North 13th Street, Philadelphia, PA 19122, USA.

ABSTRACT
The accelerating progress of research in nanomedicine and nanobiotechnology has included initiatives to develop highly-sensitive, high-throughput methods to detect biomarkers at the single-cell level. Current sensing approaches, however, typically involve integrative instrumentation that necessarily must balance sensitivity with rapidity in optimizing biomarker detection quality. We show here that laterally-confined, self-assembled monolayers of a short, double-stranded(ds)[RNA-DNA] chimera enable permanent digital detection of dsRNA-specific inputs. The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input. These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

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Detection of ethidium intercalation in a ds[RNA-DNA] matrix.Shown are the effects of ethidium bromide (EB) on the ds[RNA-DNA] matrix, as measured by fluorescence microscopy and by AFM. (a) Fluorescence analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display only nominal (background) fluorescence (shown in blue). Upon EB addition, followed by a single wash (see Methods), the fluorescence intensity (shown in red) specifically increases for the ds[RNA-DNA] matrix. The fluorescence intensity is measured in arbitrary units (au). (b) AFM analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display the same AFM imprint (shown in blue, as measured by height). Here, the initial heights for the two matrices are similar, as the inherent densities are non-identical. Upon EB addition, followed by a single wash, the topographic height (in red) specifically increases for the ds[RNA-DNA] matrix. Data are means, with standard deviations.
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f4: Detection of ethidium intercalation in a ds[RNA-DNA] matrix.Shown are the effects of ethidium bromide (EB) on the ds[RNA-DNA] matrix, as measured by fluorescence microscopy and by AFM. (a) Fluorescence analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display only nominal (background) fluorescence (shown in blue). Upon EB addition, followed by a single wash (see Methods), the fluorescence intensity (shown in red) specifically increases for the ds[RNA-DNA] matrix. The fluorescence intensity is measured in arbitrary units (au). (b) AFM analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display the same AFM imprint (shown in blue, as measured by height). Here, the initial heights for the two matrices are similar, as the inherent densities are non-identical. Upon EB addition, followed by a single wash, the topographic height (in red) specifically increases for the ds[RNA-DNA] matrix. Data are means, with standard deviations.

Mentions: The height of the ds[RNA-DNA] matrix, as well as the imprinting process, also are responsive to small molecule interactions. The binding of ethidium cation to the ds[RNA-DNA] is directly demonstrated by fluorescence enhancement (Fig. 4a) as well as increased height (Fig. 4b), both of which are consistent with intercalation of the planar cation into the surface-bound duplex structures. As additional support for an intercalative process, the height of the corresponding ss[RNA-DNA] matrix is not altered by ethidium, nor is there a significant increase in fluorescence (see right hand side of Fig. 4b). It was shown that the ethidium inhibits RNase III through dsRNA intercalation40. The addition of ethidium to the ds[RNA-DNA] matrix blocks the action of RNase III, as well as BamHI, as revealed by suppression of the imprint (HIN = HOUT = 13.1 ± 0.5 nm; data not shown).


Digital imprinting of RNA recognition and processing on a self-assembled nucleic acid matrix.

Redhu SK, Castronovo M, Nicholson AW - Sci Rep (2013)

Detection of ethidium intercalation in a ds[RNA-DNA] matrix.Shown are the effects of ethidium bromide (EB) on the ds[RNA-DNA] matrix, as measured by fluorescence microscopy and by AFM. (a) Fluorescence analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display only nominal (background) fluorescence (shown in blue). Upon EB addition, followed by a single wash (see Methods), the fluorescence intensity (shown in red) specifically increases for the ds[RNA-DNA] matrix. The fluorescence intensity is measured in arbitrary units (au). (b) AFM analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display the same AFM imprint (shown in blue, as measured by height). Here, the initial heights for the two matrices are similar, as the inherent densities are non-identical. Upon EB addition, followed by a single wash, the topographic height (in red) specifically increases for the ds[RNA-DNA] matrix. Data are means, with standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757352&req=5

f4: Detection of ethidium intercalation in a ds[RNA-DNA] matrix.Shown are the effects of ethidium bromide (EB) on the ds[RNA-DNA] matrix, as measured by fluorescence microscopy and by AFM. (a) Fluorescence analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display only nominal (background) fluorescence (shown in blue). Upon EB addition, followed by a single wash (see Methods), the fluorescence intensity (shown in red) specifically increases for the ds[RNA-DNA] matrix. The fluorescence intensity is measured in arbitrary units (au). (b) AFM analysis. Prior to EB addition, the ds- and ss[RNA-DNA] matrices display the same AFM imprint (shown in blue, as measured by height). Here, the initial heights for the two matrices are similar, as the inherent densities are non-identical. Upon EB addition, followed by a single wash, the topographic height (in red) specifically increases for the ds[RNA-DNA] matrix. Data are means, with standard deviations.
Mentions: The height of the ds[RNA-DNA] matrix, as well as the imprinting process, also are responsive to small molecule interactions. The binding of ethidium cation to the ds[RNA-DNA] is directly demonstrated by fluorescence enhancement (Fig. 4a) as well as increased height (Fig. 4b), both of which are consistent with intercalation of the planar cation into the surface-bound duplex structures. As additional support for an intercalative process, the height of the corresponding ss[RNA-DNA] matrix is not altered by ethidium, nor is there a significant increase in fluorescence (see right hand side of Fig. 4b). It was shown that the ethidium inhibits RNase III through dsRNA intercalation40. The addition of ethidium to the ds[RNA-DNA] matrix blocks the action of RNase III, as well as BamHI, as revealed by suppression of the imprint (HIN = HOUT = 13.1 ± 0.5 nm; data not shown).

Bottom Line: The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment.The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input.These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

View Article: PubMed Central - PubMed

Affiliation: Nicholson and MONALISA (MOlecularNAnotechnology for LIfe Science Applications) Laboratories of the Department of Biology, Temple University, 1901 North 13th Street, Philadelphia, PA 19122, USA.

ABSTRACT
The accelerating progress of research in nanomedicine and nanobiotechnology has included initiatives to develop highly-sensitive, high-throughput methods to detect biomarkers at the single-cell level. Current sensing approaches, however, typically involve integrative instrumentation that necessarily must balance sensitivity with rapidity in optimizing biomarker detection quality. We show here that laterally-confined, self-assembled monolayers of a short, double-stranded(ds)[RNA-DNA] chimera enable permanent digital detection of dsRNA-specific inputs. The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input. These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

Show MeSH
Related in: MedlinePlus