Detection of aptamer in tissues by qRT-PCR

RNA aptamers are rapidly gaining interest as targeting moieties for drug delivery due to their easy production and scalability, high affinity and specificity, lack of immunogenicity, and deep tissue penetration properties. Determination of the in vivo distribution of aptamers is essential to evaluate their performance and develop efficient delivery protocols. Many techniques that are currently used to perform biodistribution studies as optical imaging, magnetic resonance (MR), or nuclear medicine (as PET and SPECT) can be applied to the study of aptamers but require either the use of radioactive nucleotides or coupling the aptamers with the imaging probe de facto altering the pharmacokinetic and the biodistribution of the aptamer. These strategies often need dedicated and expensive instruments and/or the use and disposal of radioactive material. Here we describe a sensitive and safe method based on qRT-PCR for the ex vivo quantification of unmodified aptamers in tissues. 

Detection and quantification of aptamer in mouse tissues by qRT-PCR

Serena Zilio1, Paolo Serafini1,2*

1 Department of Microbiology and Immunology, Sylvester Comprehensive Cancer Center, University of Miami, Miller School of Medicine, Miami, FL 33136, USA.

2 Department of Otolaryngology, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA.

*For correspondence: pserafini@med.miami.edu

Abstract

RNA aptamers are rapidly gaining interest as targeting moieties for drug delivery due to their easy production and scalability, high affinity and specificity, lack of immunogenicity, and deep tissue penetration properties. Determination of the in vivo distribution of aptamers is essential to evaluate their performance and develop efficient delivery protocols. Many techniques that are currently used to perform biodistribution studies as optical imaging, magnetic resonance (MR), or nuclear medicine (as PET and SPECT) can be applied to the study of aptamers but require either the use of radioactive nucleotides or coupling the aptamers with the imaging probe de facto altering the pharmacokinetic and the biodistribution of the aptamer. These strategies often need dedicated and expensive instruments and/or the use and disposal of radioactive material. Here we describe a sensitive and safe method based on qRT-PCR for the ex vivo quantification of unmodified aptamers in tissues.

Keywords: Quantification, Aptamers, qRT-PCR, Tissue, Aptamer Biodistribution.

Background

RNA aptamers or “chemical antibodies” are single-stranded DNA or RNA oligonucleotides capable of binding their cognate target with high affinity and specificity because of their three-dimensional structure (Hu 2016, Parashar 2016, Qu, Yu et al. 2016, Sullenger and Nair 2016).These short chemically synthesized molecules are not immunogenic and penetrate deeply into the tissue. The fluorinated backbone of aptamers makes them resistant to RNAse degradation and incapable of triggering Toll-like Receptor (TLR) signaling (Ulrich, Martins et al. 2004, Zhu, Ye et al. 2012). Aptamers are isolated by Systematic Evolution of Ligands by Exponential enrichment (SELEX), a process in which a random library of aptamers (~1015 molecules) is partitioned using positive and negative selectors. During Cell- SELEX, aptamers are selected for internalization by receptor- or clathrin-mediated endocytosis (Farokhzad, Jon et al. 2004, Chu, Twu et al. 2006, McNamara, Andrechek et al. 2006, Magalhaes, Byrom et al. 2012, Thiel, Bair et al. 2012). This procedure allows the selected aptamer to deliver therapeutics intracellularly, allowing specific uptakes on the target cells. Last, the combinatorial use of aptamers, each binding a different epitope in the target cell, increases the overall specificity and maximizes drug delivery in the chosen tissue (De La Fuente, Zilio et al. 2020). Because of these qualities, RNA aptamers are attracting growing interest in the biomedical field for imaging and for the targeted delivery of therapeutics, allowing for higher efficacy and lower side effects.

An accurate determination of aptamer biodistribution is crucial for preclinical development and for performing IND enabling studies (Kunjachan, Ehling et al. 2015). Aptamer biodistribution studies are performed using techniques initiallylly developed for inorganic and organic compounds such as drugs, nanoparticles, or antibodies. These techniques employ, for example, immunohistochemistry, liquid scintillation counting (LSC), in vivo optical imaging, computed tomography (CT), magnetic resonance imaging (MRI),and nuclear medicineimaging techniques (Arms,Smith et al. 2018) and require aptamer modifications and specialized instruments. Additionally, these techniques are expensive, require expertise, and often involve modifying aptamer’s structure that can alter their biodistribution. Although in vivo imaging techniques (i.e. IVIS, PET, and SPECT) allow longitudinal studies and good probe quantification they are often limited by low tissue penetrance and/or spatial resolution (Arms, Smith et al. 2018). In this protocol, we described a simple, cost-effective, non-radioactive method that allows quantifying aptamers in tissues precisely. This strategy employed the aptamers’ constant region and a simple technique based on RNA extraction and qRT-PCR that most laboratories commonly use.

Materials and Reagents

1. Freshly isolated tissues from mice injected with aptamers
2. 24 and 96 well-plates (any vendor)
3. Phosphate Buffered Saline (PBS) (ThermoFisher Scientific, Gibco™, Catalog Number: 70011044)
4. Ice and ice bucket
5. RNaseZap™ (ThermoFisher Scientific, Invitrogen™, Catalog Number: AM9780)
6. Surgical Tissue Forceps - Adson Forceps, 12cm, Serrated Tips or equivalent (World Precision Instrument, Catalog Number: 14226-G)
7. Surgical scissors - Iris Scissors, 11.5cm or equivalent (WorldPrecision Instrument, CatalogNumber: 501758-G)
8. Gauze or paper towel (any vendor)
9. 1.7 mL sterile nuclease-free microcentrifuge tubes (Denville Scientific, Catalog Number: C3171)
10. Sterile Nuclease-free Barrier tips (10 µL, 200 µL, 1,000 µL) (any vendor)
11. Cordless Pestle Motor (VWR, Catalog Number: 47747-370)
12. Pestles, 1,5 ml capacity (VWR, Catalog Number: 47747-358)
13. RNA extraction reagents: TRIzol™ (ThermoFisher Scientific, Catalog Number: 15596018), Chloroform (ThermoFisher scientific Catalog Number: J67241K4), Isopropanol (Merk, Sigma- Aldrich, Catalog Number: I9030); PCR Certified Water, DNase/RNase/Endotoxin Tested (Teknova, Catalog Number: W3440); Ethyl alcohol, Pure (Merk, Sigma-Aldrich, Catalog Number: 459844)
14. MicroAmp™ Optical 96-Well Reaction Plate (ThermoFisher Scientific, Applied Biosystems™ Catalog Number: N8010560)
15. MicroAmp™ Optical Adhesive Film (ThermoFisher Scientific, Applied Biosystems™ Catalognumber: 4311971)
16. iTaq Universal SYBR Green One-Step Kit (Biorad, Catalog Number: 1725150).
17. Primers* (Stock concentration: 10 μM; Working concentration: 0.3 μM) (**) used for original PCR amplification (in the example below primers SUL5’ and SUL3’ (Table 1)
18. Purified RNA aptamers** at a known concentration (Table 1 in our example)

Notes: (*) This protocol refers to aptamers generated using the constant regions defined by the Sullenger group (Layzer and Sullenger 2007).Please use primercorresponding to the constant regionof your aptamers.

Equipment

1. -80°C freezer (anyvendor)
2. (Optional) Vaacum trap (any vendor)
3. Chemical (fume) hood (any vendor)
4. Benchtop refrigerated microcentrifuge (Eppendorf, CatalogNumber: 5417 R) or equivalent
5. Precision scale (any vendor)
6. NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (ThermoFisher Scientific, catalog number: ND-ONE-W) or equivalent
7. StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific, Applied Biosystems™, Catalog Number: 4376600) or equivalent

Software

1. Excel (Microsoft, Office suite) or equivalent
2. Sigmaplot (Systat Software) or equivalent

Procedure:

A. Tissue preparation

Note: To perform a biodistribution study in addition to the target-specific organ/s (e.g. tumor), the analysis of spleen, lung, liver, kidney, and heart is recommended. Important: use tissue from PBS injected animals as negative controls.

1. Label the needed wells of a 24 well plate with the tissue name (label two wells per tissue).
2. Add cold PBS to 24 well plate (3 ml/ well) and chill it on ice.
3. Collect desired tissues from euthanized animals and place them in the plate containing PBS.
4. Remove excess fat, nonspecific tissue, or coagulated blood from each tissue
5. Wash the tissues in PBS by moving them into a new well containing clean PBS, keep the plate on ice
6. Clean forceps, scissors, precision scale, and benchtop withRNaseZap™
7. Weigh each clean, adequately labeled 1,7 ml microcentrifuge tube on the precision scale
8. Remove the first tissue from the PBS and eliminate the excess liquid by positioning the tissue on a clean gauze (a clean paper towel can be used instead)
9. Cut a small piece of tissue, place it in the previously prepared and weighted tube, weigh the tissue and annotate the weight. Do not let the tissue dry. For optimal dissociation the piece weight should be between 40 and 100 mg
10. Immediately add two volumes/weight of TRIzol (i.e. 100 ul of TRIzol™ for 50 mg of tissue)
11. Dissociate the tissue mechanically using a Cordless Pestle Motor loaded with a new and clean disposable pestel
12. When the tissue is dissociated, add 8 volumes of TRIzol™ (i.e. 400 ul of TRIzol™ for 50 mg of tissue).
13. Incubate the tissue for 15’ at RTpestle
    Note: It is better to process each sample individually through steps 8-13 to avoid excessive drying or lysis if processing multiple tissues.
14. Transfer the sample in ice
15. (Optional – Stopping Point) Store the samples O.N. at 4°C or -80°C for up to one year

B. RNA extraction

1. If frozen, recover the samples from the -80°C freezer and let them equilibrate to RT
2. Add 0.2 ml of chloroform per 1 mL of TRIzol™ Reagent used for lysis (i.e. add 0.1 ml of chloroform for 50 mg tissue in 0.5 ml of TRIzol™ Reagent), securely cap the tube, then thoroughly mix by shaking.
3. Incubate for 2–3 minutes.
4. Centrifuge the sample for 15 minutesat 12,000 × g at 4°C. The mixtureseparates into a lower red phenol-chloroform, an interphase, and a colorless upper aqueous phase.
5. Transfer the upper aqueous phase containing the RNA to a new tube.
6. Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzol™ reagent used for lysis (i.e. 0.25 ml per a 50 mg tissue in 0.5 ml of TRIzol™ reagent).
7. Incubate for 10 minutes at 4°C.
8. Centrifuge for 10 minutes at 12,000 × g at 4°C.
9. Discard the supernatant with a micropipettor.
10. Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis.
11. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
12. Discard the supernatant with a micropipettor.
13. Air dry the RNA pellet for 5–10 minutes.
14. Resuspend the pellet in 20–50 µL of RNase-free water
15. Incubate in a heat block set at 55–60°C for 10–15 minutes
16. (Optional – Stopping Point) Proceed to the amplification or store the RNA samples at -80°C.

C. Amplification

RNA quantification and dilution

1. If frozen, recover the samples from the -80°C freezer and let them thaw in ice
2. Keep the RNA samples in ice for the whole length of the procedure
3. Perform RNA quantification using a NanoDrop™ system or similar
4. Dilute the RNA at 50 ng/µl in molecular grade nuclease-free water and keep the sample in ice.

Standard curve preparation (range 11 fM-1.5 µM)

1. Dilute the stock solution of aptamer(s) to 1.5 µM.
2. Add 15 µl of diluted aptamers to 2 wells.
3. Add 10 µl of water to 36 wells of 96-well plates.
4. Using a multichannel micropipettor, perform serial dilutions
   1. transfer 5 µl of the diluted aptamer to the first 2 wells containing the water.
   2. Mix by pipetting at least ten times and transfer 5 µl to the following 2 wells containing water.
   3. Repeat for all but the last well (water only).
5. Store the standard curve samples in ice until used

Amplification by qRT-PCR

Calculate the numberof reactions that need to be performed, consider performing triplicates (suggested) or duplicates of each sample, standard curve points, and no-template controls

1. Prepare a master mix for all reactions by adding all the requiredcomponents as detailedin Table 2; keep the master mix in ice
2. Mix the master mix thoroughly by pipetting and distribute 18 µl into each well of a MicroAmp™ Optical 96-Well Reaction Plate.
3. Add 2 µl of RNA samples, standardcurve samples, or nuclease-free water inthe appropriate wells
4. MicroAmp™ Optical Adhesive Film Gently vortex the plate
5. Spin the plate to remove any air bubbles and collect the reaction mixture in the well bottom
6. Keep the plate on ice and protected from the light
7. Program the thermal cycling protocol on the real-time PCR system as detailed in Table 3

Notes: (*) Primers and their concentration must be experimentally defined and validated according to experimental need. Primer volume, RNA volume, and water volume can be adapted according to experimental conditions. (**) The quantity of RNA per test may vary depending on the test limit of detection and aptamer distribution in different organs. An appropriate amount of starting RNA per tissue should be experimentally tested. In this protocol, 100 ng of RNA has been used per reaction. (***) Thermal cycling protocol need to be adapted according to experimental need and experimentally validated

Data analysis

Microsoft Excel or equivalent software can be used to calculate the RNA yield and analyze raw RT- qPCR amplification data. Systat Sigmaplotor equivalent softwarecan perform statistical analysis using the analysis of variance (one-way ANOVA) method (*).

1. Calculate the RNA yield obtainedfrom each tissueas the amount of RNA / gr of Tissue
2. Determine aptamer concentration in each tissue by interpolation with the calibration curve generated as indicated in the above protocol
3. Normalize the aptamer concentration in tissue by normalizing for original RNA yield
4. To ensure accuracy of the results, data should derivefrom at least two experiments with at least three biological replicates

Notes: The limit of detection of our qRT-PCR is estimated at 0.03 fmol of aptamer in 300 ng of total RNA (~3000 to 26,000 aptamer molecules/ μg of tissue). (*) The statistical method may vary depending on the experimental need

Acknowledgments

This work is supported by the DOD-BRCA idea award W81XWH-11-1-0478, the NIDDK-supported Human Islet Research Network (HIRN, RRID:SCR_014393; https://hirnetwork.org; UC4 DK 116241the NIH award, and a JDRF award to Paolo Serafini.

The original research paper for the above aptamer quantification protocol has been published in: A.De La Fuente, S.Zilio, J.Caroli, D.Van Simaeys, E.M.C. Mazza, T.A. Ince, V.Bronte, S.Bicciato, D.T. Weed, P.Serafini,  Aptamers  against mouse  and  human tumor-infiltrating  myeloid cells as reagents for targeted chemotherapy. Sci. Transl. Med.12, eaav9760 (2020). https://www.science.org/doi/10.1126/scitranslmed.aav9760

Competing interests

S.Z. and P.S. are named as inventors in nonprovisional US patent application no. 62/815,142 entitled “RNA aptamer and uses thereof,” filed by the University of Miami, regarding the use of RNA aptamer to target myeloid cells. P.S. is co-founder of wink-therapeutics for the use of RNA aptamers.

Ethics: All animal experiments were approved by the Division of Veterinary Resources and the Institutional Animal Care and Use Committee (IACUC) of the University of Miami.

References

Arms, L., D. W. Smith, J. Flynn, W. Palmer, A. Martin, A. Woldu and S. Hua (2018). "Advantages and Limitations of Current Techniques for Analyzing the Biodistribution of Nanoparticles." Front Pharmacol 9: 802.

Arms, L., D. W. Smith, J. Flynn, W. Palmer, A. Martin, A. Woldu and S. Hua (2018). "Advantages and Limitations of Current Techniques for Analyzing the Biodistribution of Nanoparticles." Frontiers in pharmacology9: 802-802.

Chu, T. C., K. Y. Twu, A. D. Ellington and M. Levy (2006). "Aptamer mediated siRNA delivery." NucleicAcids Res 34(10): e73.

De La Fuente, A., S. Zilio, J. Caroli, D. Van Simaeys, E. M. C. Mazza, T. A. Ince, V. Bronte, S. Bicciato, D. T. Weed and P. Serafini (2020). "Aptamers against mouse and human tumor-infiltrating myeloid cells as reagents for targeted chemotherapy." Sci Transl Med 12(548).

Farokhzad, O. C., S. Jon, A. Khademhosseini, T. N. Tran, D. A. Lavan and R. Langer (2004). "Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells." Cancer Research 64(21): 7668-7672.

Hu, P. P. (2016). "Recent Advances in Aptamers Targeting Immune System." Inflammation. Kunjachan, S., J. Ehling, G. Storm, F. Kiessling and T. Lammers (2015). "Noninvasive Imaging of Nanomedicines and Nanotheranostics: Principles, Progress, and Prospects." Chem Rev 115(19): 10907-10937.

Layzer, J. M. and B. A. Sullenger (2007). "Simultaneous generation of aptamers to multiple gamma- carboxyglutamic acid proteins from a focused aptamer library using DeSELEX and convergent selection." Oligonucleotides17(1): 1-11.

Magalhaes, M. L., M. Byrom, A. Yan, L. Kelly, N. Li, R. Furtado, D. Palliser, A. D. Ellington and M. Levy (2012). "A general RNA motif for cellular transfection." Mol Ther 20(3): 616-624.

McNamara, J. O., 2nd, E. R. Andrechek, Y. Wang, K. D. Viles, R. E. Rempel, E. Gilboa, B. A. Sullenger and P. H. Giangrande (2006). "Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras." Nat Biotechnol 24(8): 1005-1015.

Parashar, A. (2016). "Aptamers in Therapeutics." J Clin Diagn Res 10(6): Be01-06.

Qu, J., S. Yu, Y. Zheng, Y. Zheng, H. Yang and J. Zhang (2016). "Aptamer and its applications in neurodegenerative diseases." Cell Mol Life Sci.

Sullenger, B. A. and S. Nair (2016). "From the RNA world to the clinic." Science 352(6292): 1417- 1420.

Thiel, W. H., T. Bair, A. S. Peek, X. Liu, J. Dassie, K. R. Stockdale, M. A. Behlke, F. J. Miller, Jr. and P.

H. Giangrande (2012). "Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection." PLoS One 7(9): e43836.

Ulrich, H., A. H. Martins and J. B. Pesquero (2004). "RNA and DNA aptamers in cytomics analysis." Cytometry A 59(2): 220-231.

Zhu, G., M. Ye, M. J. Donovan, E. Song, Z. Zhao and W. Tan (2012). "Nucleic acid aptamers: an emerging frontier in cancer therapy." Chem Commun (Camb) 48(85): 10472-10480.