Team:SJTU-BioX-Shanghai/Experiments
Preparing short dsDNA by overlap PCR
Except the Gard-337 sequence with W prefix and X suffix used for testing and modulating DRIVER system, all templates used for transcription were obtained by overlap PCR. Two long primers with 20~30-mer complementarity sequence (Tm=60~63℃) were chemically synthesized to form the full-length template in the first 10 rounds of PCR.
Site-directed mutagenesis
DNA templates of aptazyme Gard-337 (with or without probes on both ends) were synthesized on plasmid pUC-57. The inactivated form (with a G12A mutation) was acquired by Hieff MutTM Multi Site-Directed Mutagenesis Kit (11004ES10, Yeason). The products were transformed into E.coli. Plasmids in monoclonal colonies were extracted (DC201-01, Vazyme) and amplified before sequencing to confirm mutation.
Electrophoresis
For analyzing aptazyme templates (~120 bp dsDNA), we used 2% agarose gel with 0.5x TBE buffer.
Gelred (TSJ002, Tsingke) was added either in gel or in loading buffer to a final concentration
of 1x. All lanes, unless noted otherwise, were run in 6x loading buffer (TSJ010, Tsingke), and
DL2000 (TSJ011, Tsingke) was used as reference. The gel was imaged with a Tanon 3500 Gel Imaging
System.
For analyzing in vitro transcription products (~100 nt ssRNA), at first we tried 1% denatured
agarose gel with 6.3% formaldehyde and MOPS buffer. The resolving power is not good. As a
result, 10% denaturing PAGE with 8 M urea and 1x TBE buffer was chosen to assess the cleavage
characteristic of RNA aptazymes. The transcription reaction was 1:1 mixed with 2x RNA loading dye
(B0363S, NEB), denatured at 65 ℃ for 10 min in a thermocycler, and chilled immediately on ice. The
gel was equilibrated by pre-electrophoresis at 160 V for 20 min in a Mini-PROTEAN ®
Electrophoresis System (Bio-Rad) in advance. Electrophoresis was performed at 240 V for 30~45
min
until the bromophenol blue dye reached the bottom of the gel. The gel was then stained with 3x
Gelred for 10~30 min before imaging with a Tanon 2500B Gel Imaging System.
Designing and preparing libraries for in vitro evolution
Libraries were designed based on sTRSV hammerhead ribozyme (i.e., replacing the loop I and II with two random loops of 5~7 bp or 30/60 bp, then adding A-rich sequences at both ends to minimal unwanted interaction). A T7 promoter was prepended to allow transcription in vitro. A total of 12 libraries were diluted to 3.33 μM each with 40 μM T7 promoter (BT88p) in TE7.8 buffer. The mixtures were incubated at 95 ℃ for 2 min and cooled at 0.5 ℃/s to allow annealing with T7 promoter. The product was used as input in the first round of DRIVER.
Detailed protocol for DRIVER (de novo rapid in vitro evolution of RNA biosensors) 1
The pipeline could be automated by applying a liquid handling system with proper modules (i.e., a thermocycler and a shaker). As we didn't have these modules, we performed DRIVER by ourselves. As we have explained in design, alternating (+) and (-) rounds were done. To improve efficiency, we prepared master mixes on ice in advance. Every reaction, once finished preparation, should be vortexed briefly and spinned down. Master mixes shouldn't be used when stored for more than 12 hours. All water used in experiments, unless noted otherwise, were treated by DEPC to remove RNase. RNAseZap® (AM9780, Thermofisher) was used to clean gloves during transcription and RT.
(+) rounds included transcription, RT, and hot start PCR, while (-) rounds need ligation reaction in addition. The PCR product of N round was used as transcription template in N+1 round. RT primer used in (+) rounds was designed to transform RNA into DNA simply, thus we used a PCR primer (BT575p). RT primers used in (-) rounds were designed to change the prefix to generate a selection pressure preferring cleaved sequences. Thus BT1508p was used to change prefix from W to Z, and BT1316p was used to change prefix from Z to W. Primers used during hot start PCR was chosen based on the product of interest (e.g., use BT1285p to amplify sequences with W prefix when changing prefix from Z to W or the prefix is W per se).
Incubate at 37 ℃ for 30 min.
Incubate at 50 ℃ for 20 min, followed by heat inactivation at 95 ℃ for 2 min. The product should be cooled to room temperature at 0.5 ℃/s.
Incubate at 37 ℃ for 15 min, followed by heat inactivation at 65 ℃ for 10 min.
Target groups for DRIVER
The target groups used in R5~R30 include:
Molecules above were dissolved in DMSO first at ~10 mg/ml before being diluted into admired
concentration in water.
The target groups used in R32~R54 includes:
Detailed protocol for CleaveSeq (NGS for measuring cleave fraction of individual sequences in a aptazyme library) 1
Hot start PCR product of (-) rounds (We used round 31 and round 55, respectively) were used as input of CleaveSeq. The concentration of dsDNA was determined by dsDNA HS Assay Kit for Qubit® (12640ES60, Yeasen) at first. Then the library was diluted ~1,000,000x to a final concentration of 7.5 fg/ml in 0.1 μg/ml salmon sperm DNA (H1060, Solarbio). The dilution was amplified under the following protocol. Thorough mixing should be guaranteed during every step described above.
The product could be used as the template of transcription. We also prepared master mix of T7 transcription, RT, and ligation for CleaveSeq as we had done in DRIVER. The hot start PCR reaction was replaced by another high-fidelity PCR.
The reference group includes 4 reference sequences at 200 pM each, which have a similar structure to
library. They were designed to normalize the abundance of each sequence in the library.
RT primer used here was designed to change the prefix from W to Z. Since BT1508p has some unwanted
bases at digestion site, Z_stop was used in RT. PCR primer groups used here were designed to add
NGS adapter on both sequences with W prefix or Z prefix. To acquire specifity, some primers (WFU,
ZFC and XRC) were blocked by a /3Spc3/ modification. 4 different primer groups with identical
function were used to distinguish samples.
Incubate at 37 ℃ for 30 min.
Incubate at 50 ℃ for 20 min, followed by heat inactivation at 95 ℃ for 2 min. The product should bei cooled to room temperature at 0.5 ℃/s.
Incubate at 37 ℃ for 15 min, followed by heat inactivation at 65 ℃ for 10 min.
Incubate at 37 ℃ for 10 min, followed by heat inactivation at 85 ℃ for 20 min.
PCR, purification and sequencing were performed by gene-sequencing company NovelBio.
For the details of the data analysis process, click
here to view our model
website.
Preparation of AuNPs-oligo conjugate
Firstly, 4 mL of 1% trisodium citrate was added into 100 mL of boiling HAuCl4 solution (0.01%). After turning red, the solution was boiled for an additional 10 min. The AuNPs solution got was stored at 4 ℃ until use 2. Before conjugation between AuNPs and oligos, add 40 μL 0.5 M TCEP into 1 ml 10 μM HS-oligo solution to activate the oligo, the mixture was incubated for 1-2 h at room temperature. Then 52 μL activated oligo solution was added into 1 ml of the AuNPs solution and shaken overnight at 4 ℃. Then the mixture was aged by adding NaCI solution of 1.5 M slowly until a final concentration of 75 mM NaCl and then incubated at 4 ℃ for 24 h. Particles were centrifuged (12×10^3 rpm, 20 min, 4 ℃) and rinsed with rinsing buffer (20 mM Na3PO4, 5% BSA, 0.25% Tween-20 and 10% sucrose) 2.
Preparation of test and control zones
For the control zone, 25 μL of 10 μM Biotin-oligo-C was mixed with 75 μL of 1 mg/mL streptavidin and incubated for 2 h at room temperature 3. The mixture was incubated at room temperature for 2 h and then centrifuged at 14,000 rpm with a centrifugal filter (cutoff of 30 kDa, Millipore) for 20 min to remove excess oligos. Then use PBS to wash the conjugate of biotin-oligo-T and streptavidin for 3 cycles. PBS was then added to the mixture up to its initial volume before centrifugation. For the test zone, 10 μL of 10 μM Biotin-oligo-C was mixed with 60 μL of 1 mg/mL streptavidin and incubated for 2 h at room temperature. The mixture was incubated at room temperature for 2 h and then centrifuged at 14,000 rpm with a centrifugal filter (cutoff of 30 kDa, Millipore) for 20 min to remove excess oligos. Then use PBS to wash the conjugate of biotin-oligo-T and streptavidin for 3 cycles. PBS was then added to the mixture up to its initial volume before centrifugation 4.
Assembly of the lateral flow strip
The strip is comprised of a sample pad (Shanghai Jie Ning biotechnology company SF-08), a conjugate pad (Shanghai Jie Ning biotechnology company G-4), NC membrane(Sartorius CN140), and an absorbent pad (Shanghai Jie Ning biotechnology company H-5076) on a backing layer. Sample pads (4 mm ×12 mm) soaked in a buffer solution (1% NaCl, 1% BSA, 0.5% Tween-20, 1% Triton X-100) and dried at 50 ℃ overnight before cutting to a specific size. Conjugate pads were dealt with the same condition and cut into 4 mm×6 mm. Then 20 μL AuNPs-oligo conjugate solution was added to the conjugate pad and then dried at 50 ℃. Solutions of Biotin-oligo-T and Biotin-oligo-C were dispensed in the test and control zones of the NC membrane and then dried at 37 ℃ for 2 h. Finally, the sample pad, conjugate pad and absorbent pad were assembled together with 2 mm overlap with each other.
Lateral flow assay procedure
Template of aptazyme was transcribed with target detected existing. And then 5 μL transcription product was mixed with 4 μL 200 mM RVC (BBI Life Sciences) and 70 μL PBS (free of RNase). The mixture was added to the sample pad. After the lateral flow assay was completed, the bands on the NC membrane were imaged with a Tanon 2500B Gel Imaging System and then analyzed by Image J.
Cleave stop & start controlled by toehold-mediated strand displacement(TMSD)
With the efficient stop oligo, the secondary structure of aptazyme will be broken and the
self-cleaving activity will be inhibited. This transcription product can be stored for a short
period (a few days) that substantial degradation won`t happen.
Then 1N μL start oligo mixed to the stopped transcription product will be enough for competing
the stop oligo down. And the refolded correctly aptazyme will show the cleaving activity again.
