TITLE: rNTPs Stock Preparation / IVT Standard Reaction
AUTHORS: Felipe  Navarro Martínez, Anibal  Arce Medina, Fernan  Federici

[DESCRIPTION]
This protocol describes the preparation of rNTPs stock solutions for their use on IVT and other molecular reactions.

The rNTPs preparation is adapted from a protocol by Berglund Lab (Goers, 2006), it contains instructions to prepare 100 millimolar (mM) stock solutions of each ribonucleotide with a final pH of 7-8 each. It is important to consider that each rNTP has a slightly different molar mass, and this value can also vary according to the degree of hydration of the reagent and provider. The molar mass value you should use to determine the resuspension volume must be the one indicated on the flask that contains each rNTP.

In our case, we have:
ATP (583,36 g/mol)
GTP (567,10 g/mol)
UTP (550,09 g/mol)
CTP (527,12 g/mol)

The second part of this protocol describes the preparation of an in vitro transcription (IVT) reaction of fluorescent RNA aptamers, like Spinach or Broccoli. In our lab, we use it to test, validate and standardize our buffers, enzymes, ribonucleotides and other reagents. Spinach and Broccoli aptamers bind to DFHBI-1T and this interaction “mimics a GFP fluorophore”, as the reaction product emits a green fluorescence that can be observed in a UV transilluminator. 

The homemade IVT-aptamer reaction was based on procedures used by the Adamala-Engelhart Laboratory (Heili et al., 2018 & Aufdembrink et al., 2020), with our own modifications.

References
- Aufdembrink, L. M., Khan, P., Gaut, N. J., Adamala, K. P., & Engelhart, A. E. (2020). Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout. Rna, 26(9), 1283-1290.
- Goers, E. (2006). Berglund: Making rNTP - OpenWetWare. OpenWetWare. https://openwetware.org/wiki/Berglund:_making_rNTP
- Heili, J. M., Gomez-Garcia, J., Gaut, N. J., Cash, B. W., Aufdembrink, L. M., Heffron, B. A., ... & Engelhart, A. E. (2018). Real-time visualization of in vitro transcription of a fluorescent RNA aptamer: an experiment for the upper-division undergraduate or first-year graduate laboratory.Journal of Chemical Education,95(10), 1867-1871.
- Paige, J. S., Wu, K. Y., & Jaffrey, S. R. (2011). RNA mimics of green fluorescent protein. Science (New York, N.Y.), 333(6042), 642–646.


[STEPS]
SECTION: rNTPs Preparation
1. Determine the mass of rNTPs that is going to be used. In this case, 100 mg of each rNTP are recommended.

SECTION: rNTPs Preparation
2. Calculate the necessary volume to obtain a 100 millimolar (mM) solution of each rNTP.

We did this by estimating how many mmoles are in 100mg and used that value to estimate the volume for each 100mM solution. In our case, we calculated:
- ATP (583,36 g/mol) --> 1714 uL
- GTP (567,10 g/mol) --> 1763 uL
- UTP (550,09 g/mol) --> 1818 uL
- CTP (527,12 g/mol) --> 1898 uL

If you are using 100 mg of each rNTP, this result should be around 1600-2100 µL


SECTION: rNTPs Preparation
3. Weight 100 mg of each rNTP separately in an 1,5 mL Eppendorf tube. Deposit them on ice.

We recommend doing this step slowly, as we experienced a lot of static between the reagents and the tube when we were doing this.


SECTION: rNTPs Preparation
4. Add 1300 µL of nuclease-free water to each tube. Vortex and deposit them on ice.

Since you need to adjust the pH of the solution, we recommend adding less water at first than the amount you will actually need so you can have a broad range of volume available for the next step.

SECTION: rNTPs Preparation
5. Measure the pH of the solution using a pH strip.
SECTION: rNTPs Preparation
6. Adjust the pH of the solution using NaOH 1 Molarity (M) and HCl 1:1 to range between 7-8

rNTPs solutions are originally very acidic (pH 2-5), so you will probably need to add only base. The HCl solution is only required if you overshoot the pH by adding too much NaOH. We recommend adding the first 200 µL of NaOH 1M and from there continue in volumes between5-20 µL until you get to the pH 7-8 range. Measure the pH each time with a pH strip and have a record of how much volume you are adding to each solution. Be careful to not go over the final volume.

SECTION: rNTPs Preparation
7. If needed, add nuclease-free water up to the final volume of each solution. Vortex each tube.
SECTION: rNTPs Preparation
8. Aliquot each tube separately, label them and store them at-20 °C until use.

SECTION: IVT Standard Reaction
9. 5X IVT buffer

Our 1X IVT buffer is composed of Tris Acetate pH 8.1 30 millimolar (mM), Magnesium glutamate 24 millimolar (mM), Potassium glutamate 90 millimolar (mM) , Spermidine 2 millimolar (mM) and DTT 1 millimolar (mM)
  Stock solutionsVolume for a 50mL solutionTris-Ac pH 8.1 500mM3 mlMg-Glutamate 500mM2.4 mlK-Glutamate 100mM4.5 mlSpermidine 1M100 uLDTT 1M50 uL
 We aliquote this 5x transcription buffer and store it at -20 °C
SECTION: IVT Standard Reaction
11. Test Reaction Protocol

The amounts shown here are for a single 20 µL reaction. 
SECTION: IVT Standard Reaction
11.1. Remember to thaw, vortex and quickly spin tubes down before opening for dispensing. It is preferable to keep all the tubes on ice when not in use. When stored, all the reagents should be at -20 °C.
 
   1x (uL)Final concentration IVT Buffer 5x41XATP 100 mM110 mMCTP 100mM110 mMUTP 100mM110 mMGTP 100mM110 mMDFHBI-1T 40 mM12 mMT7 Pol Mix (2 mg/mL)20,1 mg/mLH207DNA2Spinach or Broccoli aptamer20

SECTION: IVT Standard Reaction
11.2. Incubate the reaction at 37 °C for 180 min for downstream applications, it can also be incubated overnight, however little increase in yield is seen in our hands.
 



SECTION: IVT Standard Reaction
10. DFHBI-1T 40 mM

In case Broccoli or Spinach aptamer are used to benchmark the quality of the IVT reaction, it is necessary to prepare the following:

For a 40 millimolar (mM) solution, mix 5 mg of   with 390 µL of DMSO. Vortex, aliquot in 1,5 mL Eppendorf tubes and save at -20 °C covered with aluminium foil.