Reversibly Modified RNase H for Hot Start Nucleic Acid Detection
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Solution Overview
Problem
Current reverse transcriptase-PCR methods face challenges in accurately detecting RNA sequences due to unwanted degradation of RNA:DNA primer heteroduplexes by RNase H, which limits sensitivity and precision, especially in real-time monitoring.
Innovation Solution
A reversibly modified 'hot start' RNAse H enzyme composition with inducible activity is introduced, allowing for suppressed RNase H activity during reverse transcription and induced activity for fluorescent detection, using a thermostable RNase H domain and reversible chemical modifications such as acylation or formaldehyde treatment to control enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNase H is added to the reverse transcriptase-PCR reaction mixture, then fluorescent detection of CATACLEAVETM probes is enabled, but RNA:DNA primer heteroduplexes are degraded prior to reverse transcription
Solution Approach 1:
The RNase H enzyme is pre-added to the reaction mixture but kept inactive during the reverse transcription step through reversible modification (e.g., boronate modification). This preliminary preparation allows the enzyme to be ready for action immediately after reverse transcription completes, enabling prompt cleavage of the probe RNA portion without interfering with the reverse transcription process itself.
Solution Approach 2:
The RNase H enzyme transitions from an inactive modified state during reverse transcription to an active state afterward. The reversible modification (such as boronate modification at pH 8.0) allows dynamic control of enzyme activity, suppressing it when not needed and activating it when required for probe cleavage and fluorescent detection.
2Reliability
If RNase H activity is suppressed during reverse transcription, then RNA:DNA primer heteroduplexes are preserved, but fluorescent detection of amplification products cannot occur
Solution Approach 1:
The pH parameter is used to control RNase H activity. The enzyme is modified with boronate groups that are inactive at pH 8.0 (suppressed activity during reverse transcription) but become active at lower pH levels. This parameter change allows the same enzyme to serve dual purposes: preserving heteroduplexes during reverse transcription and enabling fluorescent detection afterward.
Solution Approach 2:
The reversible modification (boronate modification) acts as an intermediary mechanism that mediates between the conflicting requirements of preserving RNA:DNA heteroduplexes and enabling probe cleavage. The modification temporarily blocks enzyme activity when needed and can be removed or reversed when activation is required, serving as a controllable intermediary state.
3Productivity
If standard RNase H is used in real-time reverse transcriptase-PCR, then probe cleavage can occur, but sensitivity and precision are limited due to unwanted RNA degradation
Solution Approach 1:
The reversible modification pre-prevents the harmful action of RNase H (unwanted degradation of RNA:DNA primer heteroduplexes) during reverse transcription. By suppressing enzyme activity beforehand through modification, the system prevents damage before it occurs, thereby maintaining sensitivity and precision while still enabling productive probe cleavage afterward when the enzyme is activated.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enhances the sensitivity and accuracy of RNA sequence detection, enabling fast, accurate, and high-throughput analysis suitable for diagnostic applications by minimizing RNA degradation during reverse transcription and allowing for precise fluorescent detection.
Implementation Method 1
The enzyme may be reversibly modified by acylation of an amino acid of the enzyme such as lysine
Implementation Method 2
or by reaction with formaldehyde having a concentration of about 0.2 to about 1% (w/v)
Implementation Method 3
The enzyme may have a thermostable RNase H domain which can have at least 70%, 80% or 90%, 95% or 99% sequence identity to the amino acid sequence of SEQ ID NOs: 11, 12, 13 or 14
Implementation Method 4
Cleavage within the RNA portion of the annealed probe results in the separation of the fluorescent label from the quencher and a subsequent emission of fluorescence
Implementation Method 5
a RNA: DNA duplex is generated that can be cleaved by RNAse H present in the reaction mixture
Implementation Method 6
The CATACLEAVETM probe has a chimeric structure comprising an RNA sequence and a DNA sequence, and is flanked at its 5′ and 3′ ends by a detectable marker, for example FRET pair labeled DNA sequences. The proximity of the FRET pair's fluorescent label to the quencher precludes fluorescence of the intact probe
Data Source
AI summary
A reversibly modified ‘hot start’ RNase H enzyme composition is described for the improved CATACLEAVE™ probe detection of nucleic acid sequences in a test sample. A key feature of the enzyme composition is the ability to regulate the catalytic activity of the RNase H during the course of a reverse transcription-PCR cycle. Thus, RNase H activity can be initially suppressed to minimize degradation of RNA:DNA primer heteroduplexes prior to reverse transcription. After cDNA synthesis is complete, RNase H activity is induced to promote the cleavage and fluorescent detection of CATACLEAVE™ probes that anneal to target DNA sequences within the reverse transcriptase-PCR products. The inducible RNase H enzyme is amenable to high throughput applications requiring one step reverse transcriptase CATACLEAVE™ PCR in a single reaction mix.