MMLV reverse transcriptase variants
Inventors
Lee, Josephine • Marrs, Samuel • McDermott, Geoffrey • Meschi, Francesca • Montesclaros, Luz • Pfeiffer, Katherine • Shuga, Joseph Francis • Terry, Jessica Michele • Ziraldo, Solongo B.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12577545-B2
Publication Date
2026-03-17
Expiration Date
Abstract
Disclosed herein, are compositions, methods, and kits comprising engineered reverse transcription enzymes that exhibit several desired properties such as thermal stability, processive reverse transcription, non-templated base addition, and template switching ability. The engineered reverse transcription enzymes described herein demonstrate unexpectedly higher resistance to cell lysate inhibition, greater ability to capture full-length mRNA transcripts, and demonstrate improved results in small reaction volumes as compared to other engineered reverse transcription enzymes.
Core Innovation
The invention relates to an engineered reverse transcription enzyme comprising an amino acid sequence that is at least 95% identical to amino acids 26 to 671 of SEQ ID NO:3. The engineered enzyme has specified residue identities at positions corresponding to positions in SEQ ID NO:3, including K at position 69, P at position 139, N at position 200, R at position 302, K at position 306, F at position 313, and P at position 330.
Additional specified positions include an amino acid that is not L at position 435, A at position 448, G at position 449, K at position 454, N at position 524, W at position 603, and K at position 607. In the disclosed embodiments, the engineered reverse transcription enzyme includes an N-terminus truncation relative to SEQ ID NO:3 and may optionally include affinity tags and protease-cleavable tags as part of the construct.
The partial disclosure associates the engineered reverse transcription enzyme with template switching and terminal transferase activity used for cDNA synthesis and templated switching. The enzyme is described as supporting full-length mRNA capture and improved performance in the presence of cell lysate inhibition, together with enhanced thermal stability and processivity, while being used with template switching oligos and barcoded beads/oligonucleotides.
Claims Coverage
The independent claim covers one engineered reverse transcription enzyme defined by sequence identity to a specified region of SEQ ID NO:3 plus required residue identities at multiple numbered positions. The claim set centers on a single inventive concept, with the identified coverage focused on the enzyme definition and related construct refinements.
Engineered reverse transcription enzyme with SEQ ID NO:3 region identity
An engineered reverse transcription enzyme comprising an amino acid sequence that is at least 95% identical to amino acids 26 to 671 of SEQ ID NO:3.
Engineered sequence with required residue identities at SEQ ID NO:3-corresponding positions
The amino acid sequence has a K at position 69, a P at position 139, an N at position 200, an R at position 302, a K at position 306, an F at position 313, a P at position 330, an amino acid that is not L at position 435, an A at position 448, a G at position 449, a K at position 454, an N at position 524, a W at position 603, and a K at position 607, wherein the positions correspond to positions in SEQ ID NO:3.
Overall, the claim coverage is directed to an engineered reverse transcription enzyme defined by at least 95% identity to amino acids 26 to 671 of SEQ ID NO:3 and specific residue requirements at multiple numbered positions corresponding to SEQ ID NO:3.
Stated Advantages
Improves library complexity.
Reduces cell-lysate inhibition.
Reduces bias toward shorter genes and biases toward longer genes.
Increases sensitivity in full-length V(D)J transcriptional profiling.
Increases sensitivity in 3′/5′ transcriptional assays.
Increased full-length mRNA capture.
Enhanced thermal stability and processivity.
Improved cDNA synthesis/templated switching performance, including template switching and terminal transferase activity.
Documented Applications
Cell/population profiling including full-length V(D)J transcriptional profiling (TCR alpha/beta with paired V-J spanning).
3′/5′ transcriptional assays.
Cell surface protein profiling using antibody-reporter oligonucleotides.
Cell/population profiling for microbiome, infection, and drug resistance.
Use within single-cell partitioning workflows using barcoded beads/oligonucleotides and template switching oligos for templated switching and terminal transferase activity.
Use in cDNA synthesis and templated switching in small-volume reactions.
Interested in licensing this patent?