DNA-Displayed RNA Libraries for SELEX Without Reverse Transcription
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Solution Overview
Problem
The development of base-modified RNA aptamer libraries is hindered by the requirement for two different enzymes (RNA polymerase and reverse transcriptase) to tolerate modified bases, making it difficult to select RNA aptamers with enhanced serum nuclease resistance and chemical diversity using standard SELEX procedures.
Innovation Solution
A method involving DNA-displayed RNA libraries, where a ds-DNA molecule is used to capture and tether an RNA molecule, allowing it to bind to a target molecule without the need for reverse transcription, facilitating the selection of RNA aptamers with modified bases by using a 5' capture region and a rigidifier strand to maintain the RNA's secondary structure during selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If standard SELEX procedures are used for RNA aptamer selection, then RNA molecules can be selected through reverse transcription-PCR, but modified RNA bases cannot be effectively incorporated because reverse transcriptase cannot tolerate modified bases
Solution Approach 1:
The invention extracts and removes the reverse transcription step from the traditional SELEX procedure. By using DNA-displayed RNA libraries where RNA molecules are tethered to their encoding DNA sequences through capture regions, the method eliminates the need for reverse transcriptase, thereby enabling the selection of modified RNA aptamers that would otherwise be incompatible with standard procedures
Solution Approach 2:
The invention introduces an intermediary DNA capture region that mediates the connection between the RNA aptamer and its encoding DNA template. This capture region allows the RNA to be displayed and selected without requiring reverse transcription, serving as a bridge that enables modified base incorporation while maintaining the selection process
2Adaptability or versatility
If RNA libraries are generated by transcription of T7 promoter-containing random DNA library, then RNA aptamers can be selected, but the process requires both RNA polymerase and reverse transcriptase to tolerate modified bases which limits the use of modified RNA analogs
Solution Approach 1:
The invention removes the reverse transcription step from the SELEX workflow, thereby eliminating the requirement for reverse transcriptase enzyme compatibility with modified bases. This extraction of the problematic step allows unrestricted use of modified RNA analogues in the library
Solution Approach 2:
The invention changes the fundamental parameter of the selection method from reverse transcription-PCR based selection to DNA-display based selection. This parameter change shifts the enzymatic requirements from needing both RNA polymerase and reverse transcriptase to only needing RNA polymerase for transcription, thereby simplifying enzyme compatibility requirements
3Ease of manufacture
If DNA aptamers are selected using standard phosphoramidite chemistry, then DNA libraries can be synthesized and selected directly, but RNA aptamers require additional transcription and reverse transcription steps that complicate the process
Solution Approach 1:
The invention merges the RNA selection process with the DNA amplification process by displaying the RNA molecule tethered to its encoding DNA template. This merging allows the selected RNA to be directly amplified through PCR of the DNA template, combining what were previously separate transcription and amplification steps into a unified workflow
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 enables the efficient selection and amplification of RNA aptamers with high affinity for targets, such as human α-thrombin, without the need for reverse transcription, thereby overcoming the limitations of traditional SELEX methods and allowing for the incorporation of modified RNA analogues.
Implementation Method 1
a first DNA strand at least partially annealed to a first region of the RNA molecule
Implementation Method 2
allowing the second region of the RNA to bind the target molecule
Data Source
AI summary
The present invention is directed to the method for selecting an RNA molecule that binds to a target molecule and a kit for carrying out the method. This method includes: providing a pool of oligonucleotide complexes that each comprise a ds-DNA molecule and an RNA molecule, the ds-DNA molecule comprising a first DNA strand at least partially annealed to a first region of the RNA molecule, whereby a second region of the RNA molecule is free to adopt a secondary structure; exposing the pool to a target molecule and allowing the second region of the RNA to bind the target molecule; and selecting from the pool one or more oligonucleotide complexes comprising an RNA molecule having the second region bound to the target molecule.