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Generation of Chimeric DNA-RNA Structures using Engineered Replisomes in vivo

Award Number: F32GM142155
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 09/01/2021
PERIOD OF PERFORMANCE END DATE: 08/31/2024

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Generation of Chimeric DNA-RNA Structures using Engineered Replisomes in vivo - PROJECT SUMMARY/ABSTRACT The evidence of ribose in ancient catalytic machinery such as the ribosome as well as a universal set of cofactors which are derived from ribonucleic acids (RNA) has led to the hypothesis that RNA was once the dominant biopolymer in life. The transition from an RNA to DNA world raises a multitude of questions regarding how life was able to support such a dramatic change in the most fundamental information storage molecules without compromising survival. Recent studies have shown that certain randomly mutagenized strains of E. coli are in fact capable of supporting surprisingly high amounts of RNA in their genome, however it remains unclear the exact biochemical mechanisms that allow this to occur, and further studies of these strains is challenging due to inherent instability of their genomes. In this project I will be developing a novel polymerase system which could potentially limit RNA incorporation to small regions of DNA such as a plasmid. Targeting RNA incorporation in this way will allow for the first time a systematic method to study the effects of high ribose content in genes, without compromising host genomes. The proposal outlined here will focus on strategies for engineering the DNA polymerase within the multienzyme viral genome replication machinery known as the T7 replisome. In Specific Aim I, I will outline a plan to study the effects of focused mutations within the active site of T7 DNA polymerase. This will include the development and implementation of 96-well based screening method for the rapid determination of DNA polymerases with the ability to incorporate RNA permissively. Specific Aim II details a protocol for the selection of RNA permissive DNA polymerases from much larger mutant pools. A key feature of this aim is the combination of phage-display which has been used for the selection of nucleotide promiscuity in vitro along with a complementation-based selection strategy which further selects for mutant polymerases that retain functionality in vivo. We hypothesize that a novel combination of these strategies can be used to solve the inherent limitation of phage display to in vitro reaction conditions which may not accurately reflect intracellular conditions. Finally, Specific Aim III details a new approach toward the selection of mutant polymerases with the ability to incorporate RNA into replicating plasmids within a bacterial host. This method will rely on the use of alkyne labeled ribonucleotides which will provide chemical handles for the enrichment of plasmids that encode polymerases with relaxed sugar specificity. Together this project will create progress toward an orthogonal polymerase system for the construction of chimeric DNA-RNA plasmids in vivo, a task which is currently impossible using known synthetic biology tools.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = -$4,651 )
2025	2023	SCRIPPS RESEARCH INSTITUTE	10550 N TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	3	6/25/2025	NON-COMPETING CONTINUATION	-$4,651
														Subtotal = -$4,651

Issue Date FY: 2024 ( Subtotal = $0 )
2024	2023	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	3	10/11/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2023 ( Subtotal = $69,500 )
2023	2023	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	3	8/17/2023	NON-COMPETING CONTINUATION	$69,500
														Subtotal = $69,500

Issue Date FY: 2022 ( Subtotal = $67,174 )
2022	2022	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	2	9/8/2022	NON-COMPETING CONTINUATION	$67,174
														Subtotal = $67,174

Issue Date FY: 2021 ( Subtotal = $65,610 )
2021	2021	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	001	1	4/20/2021	NEW	$0
2021	2021	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	002	1	8/27/2021	NEW	$0
2021	2021	SCRIPPS RESEARCH INSTITUTE, THE	10550 NORTH TORREY PINES RD	LA JOLLA	CA	92037	SAN DIEGO	USA	Biomedical Research and Research Training	000	1	4/20/2021	NEW	$65,610
														Subtotal = $65,610

Grand Total All Awards = $197,633

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Generation of Chimeric DNA-RNA Structures using Engineered Replisomes in vivo

Award Number: F32GM142155

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 09/01/2021

PERIOD OF PERFORMANCE END DATE: 08/31/2024

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