US 12,173,334 B2
Bio-engineered hyper-functional “super” helicases
Taekjip Ha, Baltimore, MD (US); and Sinan Arslan, Urbana, IL (US)
Assigned to THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS, Urbana, IL (US)
Filed by THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS, Urbana, IL (US)
Filed on Nov. 29, 2021, as Appl. No. 17/537,419.
Application 17/537,419 is a continuation of application No. 15/526,905, granted, now 11,198,855, previously published as PCT/US2015/060693, filed on Nov. 13, 2015.
Claims priority of provisional application 62/079,183, filed on Nov. 13, 2014.
Prior Publication US 2022/0259576 A1, Aug. 18, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/14 (2006.01); C12N 9/90 (2006.01); C12P 19/34 (2006.01)
CPC C12N 9/14 (2013.01) [C12N 9/90 (2013.01); C12Y 306/04012 (2013.01); C12P 19/34 (2013.01)] 8 Claims
OG exemplary drawing
 
1. A method of catalyzing an unwinding reaction of a double-stranded DNA, comprising contacting the double-stranded DNA with a conformationally-constrained helicase
wherein the conformationally-constrained helicase is selected from the group consisting of a Rep helicase from E. coli, a UvrD helicase from E. coli, a PcrA helicase from B. stearothermophilus, or a homolog thereof;
wherein the conformationally-constrained helicase comprises a first subdomain comprising a 1A or a 1B subdomain having a first amino acid and a second subdomain comprising a 2B subdomain having a second amino acid,
wherein the first amino acid corresponds to any one of positions 84-116 or 178-196 of the helicase amino acid sequence, relative to SEQ ID NO:32;
wherein the second amino acid corresponds to any one of positions 388-411, 422-444, and 518-540 of the helicase amino acid sequence, relative to SEQ ID NO:32;
wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase;
wherein said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation, and
wherein the conformationally-constrained helicase comprises at least one degree of freedom less than a helicase that is not constrained as such.