Amplification-Free Detection of Highly Structured RNA Molecules Using SCas12aV2

Materials and reagents

Reagents

1. 10× PCR buffer (with Mg²⁺) (Beyotime, catalog number: D7223-1)

2. 10× NEBuffer r2.1 (NEB, catalog number: B6002S)

3. 1 M Tris pH 8.0, sterile (Sangon Biotech, catalog number: B548127-0500)

4. Sodium chloride (NaCl) (Sangon Biotech, catalog number: A610476-0001)

5. DTT (Sangon Biotech, catalog number: A620058-0100)

6. HOLMES-Fluo ssDNA reporter 1 (FAM) (TOLOBIO, catalog number: 31101), 5’FAM-TTATTATT-3’BHQ1

7. AsCas12a protein: Synthetic genes encoding the CRISPR-associated protein AsCas12a were cloned into the pET-28a(+) expression vector to construct plasmids for recombinant protein production. The sequence accuracy of these plasmids was verified prior to transformation into Escherichia coli BL21 (DE3) competent cells. For protein expression, a single colony was inoculated into LB broth supplemented with ampicillin (100 μg/mL) and incubated overnight. The following day, these cultures were diluted into 1 L of Terrific Broth (TB) medium to an optical density at 600 nm (OD₆₀₀) of 0.8, cooled on ice for 10 min, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and further incubated at 18 °C for 16 h. The bacterial cells were then pelleted by centrifugation and lysed in buffer A [20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM phenylmethanesulfonyl fluoride (PMSF), 5 mM β-mercaptoethanol] containing a protease inhibitor cocktail. The recombinant Cas proteins were purified using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin, followed by size-exclusion chromatography on a HiLoad^® 16/600 Superdex^® column according to a previously published protocol [5]. The purified proteins were concentrated using a 100 kDa molecular weight cutoff (MWCO) centrifugal filter, and their concentrations were determined by the Bradford assay. Subsequently, the concentrated proteins were flash-frozen in liquid nitrogen and stored at -80 °C for future use.

Solutions

1. 10× PCR buffer (with Mg²⁺) (see Recipes)

2. 10× NEBuffer r2.1 (see Recipes)

3. Storage buffer (see Recipes)

Recipes

1. 10× PCR buffer (with Mg²⁺)

100 mM Tris-HCl (pH 8.8 at 25 °C)

500 mM KCl

15 mM MgCl₂

0.8% (v/v) Nonidet P40

2. 10× NEBuffer r2.1

500 mM NaCl
100 mM Tris-HCl
100 mM MgCl₂
100 μg/mL recombinant albumin
pH 7.9 at 25 °C

3. Storage buffer

20 mM Tris-HCl

250 mM NaCl

1 mM DTT

pH 7.9 at 25 °C

Laboratory supplies

1. Pipette tips (filtered) (Thermo, catalog numbers: TF112-1000-Q, T104RS-Q, TF140-200-Q)

2. 0.2 mL PCR tubes (Thermo, catalog number: 431-MIXED-Q)

3. 1.5 mL microtubes (AXYGEN, catalog number: MCT-150-L-C)

Equipment

1. Real-time PCR system (Bio-Rad, model: CFX96 touch, or Stratagene^TM Mx3005P, Thermo, model: PF1457N)

2. Microcentrifuge (mySPIN^TM 6 Mini, Thermo, model: 75004061 or equivalent)

3. Thermal cycler (VeritiPro PCR, Thermo, model: A48141)

4. Vortex mixer (Eppendorf mixer, Eppendorf, model: 5382000074)

5. Variable volume pipettes (P10, P200, P1000)

6. Freezer (-80 °C) (Thermo, model: 905)

7. Standard laboratory equipment (analytical balance, pH meter, etc.)

Software and datasets

1. RNA secondary structure prediction software is listed as follows:

NUPACK: http://www.nupack.org/

RNAfold: http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi

Mfold: http://unafold.rna.albany.edu/?q=mfold

For the prediction of highly structured RNA regions:

1. Secondary structure prediction: Utilize NUPACK or RNAfold to identify regions with low free energy (Figure 2).

2. Target site selection: We used NUPACK to predict the secondary structures and free energy distribution of the target RNA. The results demonstrated that regions with higher free energy exhibit lower thermodynamic stability, rendering them more susceptible to disruption by activators and thus more suitable as target sites.

For the design of the dsDNA-ssDNA hybrid activators: The hybrid activator improves detection efficiency and sensitivity compared to traditional ssDNA or dsDNA activators by enabling stronger and more stable interactions between the RNA substrate and the Cas12a active site. The design principle is outlined in the following:

1. A double-stranded DNA region: 12–16 bp for scaffold binding (Figure 3).

2. PAM sequence: TTTN (N = A, C, or G).

3. A single-stranded DNA region: 20 nt complementary to the target.

Procedure