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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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Related in: MedlinePlus

Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix.(a) The final heights (HOUT) of six separate Inputs are dependent upon the initial height (HIN) of the ds[RNA-DNA] matrix.  Input 1 (with RNase III). Input 2 (with E110A).  Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg2+).  Input 1+ (with RNase III and BamHI).  Input 2+ (with E110A and BamHI).  Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: HOUT = HIN). BamHI gains full access to its site in combination with RNase III (Output 1+) as HOUT 1+ ≪ HOUT 1, while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as HOUT 2+ ~ HIN. BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, HOUT 3+ > HOUT 1 for relatively dense matrices (HIN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).
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f3: Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix.(a) The final heights (HOUT) of six separate Inputs are dependent upon the initial height (HIN) of the ds[RNA-DNA] matrix. Input 1 (with RNase III). Input 2 (with E110A). Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg2+). Input 1+ (with RNase III and BamHI). Input 2+ (with E110A and BamHI). Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: HOUT = HIN). BamHI gains full access to its site in combination with RNase III (Output 1+) as HOUT 1+ ≪ HOUT 1, while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as HOUT 2+ ~ HIN. BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, HOUT 3+ > HOUT 1 for relatively dense matrices (HIN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).

Mentions: The height of a dsDNA monolayer is proportional to its density2122. The question thus arises whether the outputs observed in this study also exhibit a similar dependence on monolayer density. Fig. 3a shows the output from Input 1 as a function of ds[RNA-DNA] matrix density, as qualitatively gauged by initial height (HIN). Specifically, an HIN of >11.5 nm corresponds to the maximum height calculated for a near-vertical orientation of the molecules (see also Discussion), reflecting the near-maximal density of the ds[RNA-DNA] matrix. Output 1 (Fig. 3a, solid red triangles) shows that the change in height of the ds[RNA-DNA] matrix linearly correlates with HIN, indicating that the RNase III cleavage site remains accessible, regardless of density. When BamHI is included, the results (Output 1+; Fig. 3a, open triangles) show that the dsDNA segment is accessible to BamHI in the presence of RNase III, also regardless of density. A requirement for the prior action of RNase III for the BamHI reaction is likely, since the same results are obtained if the ds[RNA-DNA] matrix is pre-treated with RNase III, then incubated with BamHI following removal of RNase III (data not shown). In contrast, the action of BamHI is inefficient when the relatively dense ds[RNA-DNA] matrix (HIN > 10 nm) does not receive prior treatment with RNase III (Output 3+, Fig. 2d; open diamonds). Finally, BamHI action is fully suppressed in the presence of the E110A mutant, regardless of matrix density (Output 2+, open circles). The results shown in Figs. 2 and 3 indicate that BamHI action exhibits a dependence on RNase III catalytic action (+RNase III) or RNase III binding (+E110A) that occurs in the dsRNA segment. Thus, BamHI can effectively capture either a catalytic or noncatalytic RNA-protein interaction by generating a specific, permanent, AFM-readable matrix imprint.


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

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

Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix.(a) The final heights (HOUT) of six separate Inputs are dependent upon the initial height (HIN) of the ds[RNA-DNA] matrix.  Input 1 (with RNase III). Input 2 (with E110A).  Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg2+).  Input 1+ (with RNase III and BamHI).  Input 2+ (with E110A and BamHI).  Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: HOUT = HIN). BamHI gains full access to its site in combination with RNase III (Output 1+) as HOUT 1+ ≪ HOUT 1, while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as HOUT 2+ ~ HIN. BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, HOUT 3+ > HOUT 1 for relatively dense matrices (HIN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: Density-dependent steric regulation of imprinting a ds[RNA-DNA] matrix.(a) The final heights (HOUT) of six separate Inputs are dependent upon the initial height (HIN) of the ds[RNA-DNA] matrix. Input 1 (with RNase III). Input 2 (with E110A). Input 3 (controls, either lacking RNase III or with RNase III without the catalytic cofactor, Mg2+). Input 1+ (with RNase III and BamHI). Input 2+ (with E110A and BamHI). Input 3+ (with BamHI alone). All dashed lines in (a) relate the data points to a linear regression. The data for Output 1 show that RNase III can process the dsRNA segment regardless of ds[RNA-DNA] density, which is related to the initial height (see schematic representation on top). Outputs 2 and 3 are consistent with an unaltered ds[RNA-DNA] chimera (represented by the solid diagonal line: HOUT = HIN). BamHI gains full access to its site in combination with RNase III (Output 1+) as HOUT 1+ ≪ HOUT 1, while it is essentially completely blocked in combination with the E110A mutant (Output 2+) as HOUT 2+ ~ HIN. BamHI restriction enzyme efficiency acting alone (Input 3+) must be lower than that of RNase III alone (Input 1), as the height of an matrix consisting of ds[RNA-DNA] molecules cleaved by BamHI would be lower than the height of an matrix cleaved by RNase III, and, in contrast, HOUT 3+ > HOUT 1 for relatively dense matrices (HIN > 10 nm). Data are means, and include standard deviations. (b) Schematic depiction of the effect of different inputs on a highly dense ds[RNA-DNA] matrix, including a steric hindrance-based model that shows how the ‘imprint’ (Output n+) is a step (i.e. digital) function of Input n+ (n = 1,3), as shown in (a).
Mentions: The height of a dsDNA monolayer is proportional to its density2122. The question thus arises whether the outputs observed in this study also exhibit a similar dependence on monolayer density. Fig. 3a shows the output from Input 1 as a function of ds[RNA-DNA] matrix density, as qualitatively gauged by initial height (HIN). Specifically, an HIN of >11.5 nm corresponds to the maximum height calculated for a near-vertical orientation of the molecules (see also Discussion), reflecting the near-maximal density of the ds[RNA-DNA] matrix. Output 1 (Fig. 3a, solid red triangles) shows that the change in height of the ds[RNA-DNA] matrix linearly correlates with HIN, indicating that the RNase III cleavage site remains accessible, regardless of density. When BamHI is included, the results (Output 1+; Fig. 3a, open triangles) show that the dsDNA segment is accessible to BamHI in the presence of RNase III, also regardless of density. A requirement for the prior action of RNase III for the BamHI reaction is likely, since the same results are obtained if the ds[RNA-DNA] matrix is pre-treated with RNase III, then incubated with BamHI following removal of RNase III (data not shown). In contrast, the action of BamHI is inefficient when the relatively dense ds[RNA-DNA] matrix (HIN > 10 nm) does not receive prior treatment with RNase III (Output 3+, Fig. 2d; open diamonds). Finally, BamHI action is fully suppressed in the presence of the E110A mutant, regardless of matrix density (Output 2+, open circles). The results shown in Figs. 2 and 3 indicate that BamHI action exhibits a dependence on RNase III catalytic action (+RNase III) or RNase III binding (+E110A) that occurs in the dsRNA segment. Thus, BamHI can effectively capture either a catalytic or noncatalytic RNA-protein interaction by generating a specific, permanent, AFM-readable matrix imprint.

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