The University of Texas System

The Ohio State University

Georg Mohr

Alan M Lambowitz

Steven Zimmerly

Sabine Mohr

Clifford James Beall

Eman Ghanem

Huatao Guo

Allen M Lambowitz

Alfred M Lentzsch

Roland Saldanha

Manabu Matsuura

Jennifer L Stamos

Seung Kuk Park

Huatao Gau

Jiam Yang

A crystal structure of a Non-LTR-retroelement reverse transcriptase and methods of using the same to identify enzymes with improved activity are provided. Mutant reverse transcriptase enzymes and methods of using the same are also provided.

Non-LTR-retroelement reverse transcriptase and uses thereof

Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Stabilized reverse transcriptase fusion proteins

Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse temperature. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

Methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double-stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate to permit the nucleotide integrase to cleave the top strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that interacts with a first sequence element and a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences for hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

Methods for cleaving single-stranded and double-stranded DNA substrates with nucleotide integrase

The present invention provides new methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double- stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on the one strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate under conditions that permit the nucleotide integrase to cleave the one strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that is capable of binding to a first sequence element and to a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences that are capable of hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate for a time and at a temperature sufficient to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

Methods for cleaving DNA with nucleotide integrases

Methods for preparing nucleotide integrases are provided. The nucleotide integrases are prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as 'exogenous RNA', with a group II intron-encoded protein. The exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises a group II intron sequence. In one embodiment, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule that comprises at least the open reading frame sequence of a group II intron and then expressing the open reading frame sequence in the host cell. The DNA molecule may comprise the open reading frame sequence operably linked to a promoter, preferably an inducible promoter. Thereafter, the cell is fractionated and the protein is recovered and combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity. In another embodiment, the DNA molecule comprise a group II intron sequence that encodes both a group II intron RNA as well as a group II intron encoded protein. The DNA molecule is then expressed in the host cell to provide RNP particles that comprise the group II intron-encoded protein bound to the group II intron RNA. Thereafter, the RNP particles comprising the group II intron-encoded protein and the group II intron RNA are isolated from the cell and treated with a nuclease to remove the RNA and to provide the group II-intron encoded protein. The group II intron-encoded protein is then combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity.

Methods of making an RNP particle having nucleotide integrase activity

The present invention provides new, improved, and easily manipulable methods for making nucleotide integrases. In one embodiment, the nucleotide integrase is prepared by introducing a DNA molecule which comprises a group II intron DNA sequence into a host cell. The group II intron DNA sequence is then expressed in the host cell such that RNP particles having nucleotide integrase activity are formed in the cell. Such RNP particles comprise an exiced group II intron RNA encoded by the introduced DNA molecule and a group II intron-encoded protein encoded by the introduced DNA molecule. Thereafter, the nucleotide integrase is isolated from the cell. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as 'exogenous RNA', with a group II intron-encoded protein. In another embodiment, the nucleotide integrase is prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as 'exogenous RNA', with an RNA-protein complex which comprises a group II intron-encoded protein. Preferably, the exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises the group II intron sequence. Preferably, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule which comprises the open reading frame sequence of a group II intron, and then expressing the the open reading sequence in the host cell such that the group II intron-encoded protein encoded by the open reading frame sequence is formed in the cell. Preferably, the RNA-protein complex is made by introducing into a host cell a DNA molecule comprising a group II intron DNA sequence which encodes a splicing-defective group II intron RNA. The present invention also relates to a nucleotide integrase and an improved method for making RNA-protein complexes for use in preparing nucleotide integrases in vitro.

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Austin, TX, United States of America

Georg Mohr