Journal: bioRxiv

Article Title: Multifaceted mRNA analysis using programmed RNA cleavage by Mucilaginibacter paludis Argonaute

doi: 10.1101/2025.07.01.662632

Figure Lengend Snippet: (A) pSG90 IVT RNA (∼1.7 kb) was subject to MpaAgo cleavage with single complementary DNA guides in ThermoPol® reaction buffer at 50°C for 15 minutes. The substrate:Ago molar ratio was 1:5, and the Ago:guide molar ratio was 1:5. 16 nt guides were 5’ phosphorylated and were designed for MpaAgo to cleave 100 nt apart from each other. Cleavage products were analyzed on a denaturing 6% TBE-urea gel stained with SYBR Gold. Guide identities and cleavage efficiencies (%) are indicated above the corresponding lanes. (B) Fluc IVT RNA (∼1.8 kb) was subject to MpaAgo cleavage with single complementary DNA guides. Cleavage reactions were performed under the same conditions as in (A). 16 nt guides for Fluc RNA were also 5’ phosphorylated but were designed for MpaAgo to cleave 72 nt apart from each other. Cleavage products were analyzed on a denaturing 6% TBE-urea gel stained with SYBR Gold. Guide identities and cleavage efficiencies (%) were indicated above the corresponding lanes.

Article Snippet: 200 ng of Fluc RNA (∼1.8 kb, ∼0.37 pmol), a shortened version of the pTsin saRNA (pSG90; ∼1.7 kb, ∼0.37 pmol), or Epo RNA with a modified sequence (pSG95; ∼0.7 kb, ∼0.86 pmol), all synthesized using in vitro transcription (IVT) (New England Biolabs, #E2040) according to the manufacturer’s instructions, were cleaved with MpaAgo in a 10 μL reaction at 50°C for either 15 minutes (single and double cleavages, 1:5 substrate: Ago molar ratio) or 30 minutes (multiplexing, 1:10 substrate: Ago ratio) in 1x ThermoPol® reaction buffer (New England Biolabs #B9004S).

Techniques: Staining