Approach:
The specific aim of this Phase I project is to establish the technical merit and feasibility of detection of HCV RNA using an innovative assay called TARA. The overall work principles and design of the assays are shown in Figure 1. We will use peptide nucleic acid (PNA)-based probes to investigate in detail the signaling power and the selectivity of a transfer reaction (Figure 2). PNA is a chemically and biologically stable DNA analogue that combines an increased affinity towards DNA (RNA) with a hybridization that is highly sequence selective [28]. Due to modifications in its backbone, it does not degrade in the presence of proteases such as Proteinase K. Sequence-specific PNA oligonucleotides are labeled with antigen (biotin, or …show more content…
We have preliminary data for TARA that shows the technique is sufficiently sensitive to detect 0.005 fmoles of synthetic RNA template by eye and/or a smartphone scanner using transfer reaction molecular amplification and GNP signal amplification alone on a lateral flow strip (See Figure 3). Moreover, there was no background signal from no template control (non-templated transfer, Figure 3A) or a single base mismatch RNA template (Figure 3C). The reaction conditions are 150 nM A probe, 100 nM G probe conjugated with GNP incubated at room temperature for 30 minutes in the CLBuffer. Detection is linear with a correlation coefficient r2 of 0.93 at this low detection range. The test line bands were quantitated by scanning in grayscale with VueScan App (Harrick Software) on iphone 6 to a jpeg file which was opened and quantitated in Adobe Photoshop v.CS5.1. In order to lower costs, we propose the use of …show more content…
Using Primer Express version 2 (Applied Biosystems) software, CrossLife Technologies (CLT) will design the PNA probe sets targeting several highly conserved regions within the HCV. In Phase 1, only probes to HCV will be synthesized and tested. The other probes will be synthesized and tested in Phase 2 when we develop the multiplex detection test for HCV, HBV, HDV, and HEV. PNAs can be easily synthesized and functionalized, are more stable, and are more responsive to point-mutations than their DNA counterpart. Several PNA probe combinations will be evaluated experimentally to determine the most efficient combination. For each target RNA, 2 PNA probe pairs [G-probe with C-terminus FITC and A-probe (antigen, or biotin or antibody)] will be designed and evaluated. Probe design will be performed via Primer Express version two software. The software will create probe sets designed. Probe hybridization sequences will be evaluated by BLAST to select regions conserved across multiple transcript variants. For initial design, target complexity and accessibility will be evaluated using Visual OMP (DNA Software Inc., Ann Arbor, MI). We have identified highly conserved sequences that match our specifications for detecting
The specific aim of this Phase I project is to establish the technical merit and feasibility of detection of HCV RNA using an innovative assay called TARA. The overall work principles and design of the assays are shown in Figure 1. We will use peptide nucleic acid (PNA)-based probes to investigate in detail the signaling power and the selectivity of a transfer reaction (Figure 2). PNA is a chemically and biologically stable DNA analogue that combines an increased affinity towards DNA (RNA) with a hybridization that is highly sequence selective [28]. Due to modifications in its backbone, it does not degrade in the presence of proteases such as Proteinase K. Sequence-specific PNA oligonucleotides are labeled with antigen (biotin, or …show more content…
We have preliminary data for TARA that shows the technique is sufficiently sensitive to detect 0.005 fmoles of synthetic RNA template by eye and/or a smartphone scanner using transfer reaction molecular amplification and GNP signal amplification alone on a lateral flow strip (See Figure 3). Moreover, there was no background signal from no template control (non-templated transfer, Figure 3A) or a single base mismatch RNA template (Figure 3C). The reaction conditions are 150 nM A probe, 100 nM G probe conjugated with GNP incubated at room temperature for 30 minutes in the CLBuffer. Detection is linear with a correlation coefficient r2 of 0.93 at this low detection range. The test line bands were quantitated by scanning in grayscale with VueScan App (Harrick Software) on iphone 6 to a jpeg file which was opened and quantitated in Adobe Photoshop v.CS5.1. In order to lower costs, we propose the use of …show more content…
Using Primer Express version 2 (Applied Biosystems) software, CrossLife Technologies (CLT) will design the PNA probe sets targeting several highly conserved regions within the HCV. In Phase 1, only probes to HCV will be synthesized and tested. The other probes will be synthesized and tested in Phase 2 when we develop the multiplex detection test for HCV, HBV, HDV, and HEV. PNAs can be easily synthesized and functionalized, are more stable, and are more responsive to point-mutations than their DNA counterpart. Several PNA probe combinations will be evaluated experimentally to determine the most efficient combination. For each target RNA, 2 PNA probe pairs [G-probe with C-terminus FITC and A-probe (antigen, or biotin or antibody)] will be designed and evaluated. Probe design will be performed via Primer Express version two software. The software will create probe sets designed. Probe hybridization sequences will be evaluated by BLAST to select regions conserved across multiple transcript variants. For initial design, target complexity and accessibility will be evaluated using Visual OMP (DNA Software Inc., Ann Arbor, MI). We have identified highly conserved sequences that match our specifications for detecting