Enzymatic Synthesis of High-Density RNA Microarrays and RNA Aptamer Applications

The objective of this project is to develop a powerful new method for synthesizing high-density microarrays of long RNA oligonucleotides for RNA molecular cell biology and RNA aptamer biosensors applications, and to demonstrate their use for understanding the specificities and affinities of RNA-binding molecules. The RNA microarrays will be synthesized enzymatically from template microarrays of long DNA oligonucleotides (60 to 100mers). Conventional in situ synthesis is used to make arrays of DNA template oligonucleotides, which are then enzymatically double-stranded with RNA via primer extension with the TGK DNA polymerase. The TGK polymerase has been engineered with two key mutations that allow it to efficiently transcribe DNA to RNA via primer extension using ribonucleotide triphosphates. The primers are covalently bound to the microarray surface so that the RNA remains attached to the point of synthesis after enzymatic degradation of the DNA template. This method has been shown to be efficient and effective in preliminary experiments. The proposed experiments will help to develop several alternative primer attachment methods, as well as several enzymatic pathways to template degradation, with the objective of developing a practical and robust method for converting DNA microarrays into RNA microarrays. For this project, DNA microarrays with up to 780 thousand unique sequences will be made using Maskless Array Synthesis (MAS) and converted to RNA. The RNA microarrays will be used to comprehensively identify and understand the sequence and structural features of the cyclic-di-GMP riboswitch aptamer of Vibrio cholera riboswitch involved in determining binding affinity.

The objective of this project was to develop a powerful new method for synthesizing high-density microarrays of long RNA oligonucleotides for RNA molecular cell biology and RNA aptamer biosensors applications, and to demonstrate their use for understanding the specificities and affinities of RNA-binding molecules. The results of the project allow RNA microarrays to be synthesized enzymatically from template microarrays of long DNA oligonucleotides (60 to 100mers). Conventional microarray synthesis is used to make arrays of DNA template oligonucleotides, to which RNA or 2OMeRNA primers are hybridized and then permanently attached using photochemical crosslinking reactions. These microarrays are then enzymatically double-stranded with RNA via primer extension with the T7 RNA polymerase. For this project, DNA microarrays with up to 780 thousand unique sequences will be made using Maskless Array Synthesis (MAS) and then converted to RNA. The same process will also allow customized commercial DNA microarrays to be converted to RNA. The RNA microarrays will be used to study bioaffinity interactions specific to RNA, such as RNA binding proteins, riboswitches and RNA processing enzymes.