Field of the Invention

The present invention relates to aptamers and particularly, although not exclusively, to aptamers that bind elF4e and to the formulation and application of aptamers as a spray, aerosol, vapour, or mist.

Background to the Invention

The leading treatment option for cancer, when possible, is surgical resection followed by chemo/radio therapy. This normally gives the best chance of survival and is the most common therapeutic option taken before metastasis of the tumor. There is however a lag time between surgery and the start of chemo/radiotherapy because the patient needs time to recover from the surgery. In that short span of time, residual cancer cells can begin to divide and colonize the lesion left behind by the surgery.

Mochizuki et al (High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation. RNA 2005 11 :77-89) and JP03940097B2 disclose two RNA aptamers, with high affinity for mammalian elF4E by in vitro RNA selection-amplification. Summary of the Invention

In one aspect of the present invention a DNA aptamer is provided that binds elF4e, preferably with high affinity and/or high specificity.

In some aspects of the present invention an aptamer having a nucleic acid sequence comprising or consisting of one of SEQ ID NOs 1 to 69, or one of the sequences shown in Figure 2a, Figure 7, Figure 8 or Figure 12 is provided. In some embodiments, an aptamer having a nucleic acid sequence comprising or consisting of one of SEQ ID NOs 43 to 48 is provided. In some embodiments, an aptamer having a nucleic acid sequence comprising or consisting of one of SEQ ID NOs 1 to 22 is provided. In some

embodiments, an aptamer having a nucleic acid sequence comprising or consisting of one of: SEQ ID NOs: 23 to 29; SEQ ID NOs: 30 to 33; SEQ ID NO: 34; SEQ ID NOs: 35 to 42; SEQ ID NOs: 49 to 51 ; SEQ ID NOs: 52 to 65; SEQ ID NO: 66; SEQ ID NO: 67; SEQ ID NO: 68; or SEQ ID NO: 69, is provided. In some embodiments an aptamer may contain one of SEQ ID NOs 1 to 69 as well as additional nucleic acid sequences at one or both of the 5' or 3' ends. For example, in some embodiments an aptamer may further comprise, or further consist of, the sequence CTTCCGATCT at the 5' end and/or may further comprise, or consist of, the sequence AGATCGGAAG at the 3'end.

In some preferred embodiments the aptamer is a deoxyribonucleic acid. In other embodiments the aptamer may be a ribonucleic acid. The aptamer is preferably single stranded.

In some embodiments the aptamer may have free ends. For example, the 3' and 5' ends may not be ligated to form a loop, although they may be conjugated to other molecules or otherwise modified, e.g. by 3'-capping, for example with an inverted thymidine. The aptamer may adopt a tertiary structure such as a hairpin loop. In some embodiments the aptamer is looped. For example, the 5' and 3' ends of the nucleic acid are covalently bonded, e.g. by ligation, to form a loop not having any free ends and which may still be single stranded.

In some embodiments an aptamer according to the present invention may dimerise either with an identical aptamer or a different aptamer to form a homo- or hetero- dimer, respectively. Whilst the dimers may interact by hydrogen bonding, optionally they may not undergo Watson-Crick base pairing and may retain a single stranded form.

Aptamers according to the present invention may contain one or more bases that are chemically modified. In some embodiments, each base of a given type (e.g. A, T, C, G) may contain the same chemical modification. Aptamers according to the present invention may contain one or more nucleotides that are chemically modified at the 2' position of ribose. In some embodiments, each ribose contains the same chemical modification. In some other embodiments the ribose of certain nucleotides (e.g. A, T, C, G) may be independently modified. Such modifications may include O-methyl modification (2'-OMe), Fluoride modification (2'-F) or amine modification (2-NH ₂ ).

In another aspect of the present invention a pharmaceutical composition or medicament is provided, the pharmaceutical composition or medicament comprising an aptamer as described herein and a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutical composition or medicament may be formulated as a spray, aerosol, vapour, or mist. This may involve formulation as a liquid or powder suitable for delivery by way of a spray applicator, nebuliser, atomiser or vaporiser. Accordingly, in another aspect of the present invention a drug delivery device is provided comprising a pharmaceutical composition or medicament as described herein, the device having an applicator operable to deliver the pharmaceutical composition or medicament as a spray, aerosol, vapour or mist. The device may comprise a housing for the aptamer, e.g. for a liquid or powder formulation, and an applicator for formation of the spray, aerosol, vapour or mist. The applicator may comprise a nebuliser, atomiser or vaporiser.

In another aspect of the present invention the aptamer, pharmaceutical composition or medicament described herein is provided for use in therapy or in a method of medical treatment. Such use may be for the treatment of cancer. In another aspect of the present invention there is provided the use of an aptamer as described herein in the manufacture of a pharmaceutical composition or medicament for the treatment of disease. The disease may be cancer.

In another aspect of the present invention a method of treating a disease in a patient in need of such treatment is provided, the method comprising administering an aptamer as described herein to the patient, thereby treating the disease. The disease may be cancer.

Administration of aptamer, pharmaceutical composition or medicament may be in the form of a spray, aerosol, vapour, or mist. Administration of aptamer, pharmaceutical composition or medicament may be to tissue in need of treatment, e.g. cancerous tissue, or to a site of surgical resection of such tissue. In another aspect of the present invention a pharmaceutical composition or medicament comprising an aptamer and a pharmaceutically acceptable carrier, excipient or diluent is provided, wherein the pharmaceutical composition or medicament is formulated as a spray, aerosol, vapour, or mist. This may involve formulation as a liquid or powder suitable for delivery by way of a spray applicator, nebuliser, atomiser or vaporiser. In another aspect of the present invention a drug delivery device comprising a pharmaceutical composition or medicament is provided comprising an aptamer, the device having an applicator operable to deliver the pharmaceutical composition or medicament as a spray, aerosol, vapour or mist. The device may comprise a housing for the aptamer, e.g. for a liquid or powder formulation, and an applicator/nozzle for formation of the spray, aerosol, vapour or mist. The applicator may comprise a nebuliser, atomiser or vaporiser. In another aspect of the present invention there is provided an aptamer for use in a method of medical treatment, wherein the method comprises administration of the aptamer as a spray, aerosol, vapour or mist. In another aspect of the present invention there is provided the use of an aptamer in the manufacture of a pharmaceutical composition or medicament for the treatment of a disease in a subject, wherein the treatment involves administration of the aptamer as a spray, aerosol, vapour or mist. In another aspect of the present invention a method of treating a disease in a patient in need of such treatment is provided, the method comprising administering an aptamer to the patient as a spray, aerosol, vapour or mist, thereby treating the disease. In some embodiments the disease being treated is cancer. In some embodiments administration of aptamer, pharmaceutical composition or medicament may be to tissue in need of treatment, e.g. cancerous tissue, or to a site of surgical resection of such tissue.

Description

The inventors have developed an aptamer that is able to inhibit tumour cell proliferation by blocking elF4e activity. Unlike chemotherapeutic and radiotherapeutic agents that also kill dividing healthy cells, inhibition of elF4e inhibits cell proliferation and thus allows healthy cells to survive.

Aptamers

Aptamers, also called nucleic acid ligands, are nucleic acid molecules characterised by the ability to bind to a target molecule with high specificity and high affinity. Almost every aptamer identified to date is a non-naturally occurring molecule.

Aptamers to a given target, including those of the present invention, may be identified and/or produced by the method of Systematic Evolution of Ligands by Exponential enrichment (SELEX™). Aptamers and SELEX are described in Tuerk and Gold

(Systematic evolution of ligands by exponential enrichment: RNA ligands to

bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505-10) and in W091/19813.

Aptamers may be DNA or RNA molecules and may be single stranded or double stranded. The aptamer may comprise chemically modified nucleic acids, for example in which the sugar and/or phosphate and/or base is chemically modified. Such modifications may improve the stability of the aptamer or make the aptamer more resistant to degradation and may include modification at the 2' position of ribose.

Aptamers may be synthesised by methods which are well known to the skilled person. For example, aptamers may be chemically synthesised, e.g. on a solid support.

Solid phase synthesis may use phosphoramidite chemistry. Briefly, a solid supported nucleotide is detritylated, then coupled with a suitably activated nucleoside

phosphoramidite to form a phosphite triester linkage. Capping may then occur, followed by oxidation of the phosphite triester with an oxidant, typically iodine. The cycle may then be repeated to assemble the aptamer.

Aptamers can be thought of as the nucleic acid equivalent of monoclonal antibodies and often have Kd's in the nM or pM range, e.g. less than one of 500nM, 100nM, 50nM, 10nM, 1 nM, 500pM, 100pM. As with monoclonal antibodies, they may be useful in virtually any situation in which target binding is required, including use in therapeutic and diagnostic applications, in vitro or in vivo. In vitro diagnostic applications may include use in detecting the presence or absence of a target molecule. Aptamers according to the present invention may be provided in purified or isolated form. Aptamers according to the present invention may be formulated as a pharmaceutical composition or medicament.

Aptamers according to the present invention may optionally have a minimum length of one of 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides

Aptamers according to the present invention may optionally have a maximum length of one of 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides

Aptamers according to the present invention may optionally have a length of one of 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides. Aptamers according to the present invention may have a degree of primary sequence identity with one of SEQ ID NOs 1 to 69, or one of the sequences shown in Figure 2a, Figure 7, Figure 8 or Figure 12 that is at least one of 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

Aptamers according to the present invention may bind elF4e, preferably mammalian or human elF4e, with high affinity and specificity. They may also act as inhibitors of elF4e activity, e.g. may inhibit translation and/or transcription.

Aptamers according to the present invention may also inhibit proliferation of cells in vitro or in vivo. This may involve inhibition of cancer/tumor cell proliferation. This may be a cytostatic effect, but may also be a cytotoxic effect. As such, aptamers according to the present invention are provided for use in methods of medical treatment where inhibition of cell proliferation is useful for treatment of a disease.

Attenuation of elF4e has also been reported to increase tumor cell radiosensitivity (Hayman et al., Cancer Res; 72(9); 2362-72. 2012 AACR). Therefore, aptamers according to the present invention may be used in a method of treating cancer wherein the method involves administration of the aptamer to the subject, as described herein, followed by treatment with ionising radiation (e.g. radiotherapy using X-rays or γ-rays). The treatment regime may indicate one or more of: the dose of each aptamer and/or radiation; the time interval between ionising radiation treatments; the length of each treatment; the number and nature of any treatment holidays, if any etc.

Methods according to the present invention may be performed in vitro or in vivo. The term "in vitro" is intended to encompass experiments with materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term "in vivo" is intended to encompass experiments and procedures with intact multi-cellular organisms.

The aptamers of the present invention may include chemical modifications as described herein such as a chemical substitution at a sugar position, a phosphate position, and/or a base position of the nucleic acid including, for example., incorporation of a modified nucleotide, incorporation of a capping moiety (e.g. 3' capping), conjugation to a high molecular weight, non-immunogenic compound (e.g. polyethylene glycol (PEG)), conjugation to a lipophilic compound, substitutions in the phosphate backbone. Base modifications may include 5-position pyrimidine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo- or 5-iodo-uracil, backbone modifications. Sugar modifications may include 2'-amine nucleotides (2'-NH2), 2'-fluoro nucleotides (2'-F), and 2'-0-methyl (2'-OMe) nucleotides.

Cancer

A cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the colon, pancreas, lung, breast, uterus, stomach, kidney, testis, central nervous system (including the brain), peripheral nervous system, skin, blood or lymph.

In some embodiments, the cancer may be a melanoma, neuroblastoma, lung adenocarcinoma, osteosarcoma, or glioma. Subjects

The subject to be treated may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic uses may be in human or animals (veterinary use).

Formulation and Administration

Medicaments and pharmaceutical compositions according to aspects of the present invention may be formulated for administration by a number of routes. The medicaments and compositions may be formulated in fluid or solid (including powder) form.

In some preferred embodiments the aptamer is formulated for administration as a spray, aerosol, vapour or mist useful to administer the aptamer to live cells in situ. This may comprise providing the aptamer in liquid, e.g. liquid suspension, or powder form and forming a spray, aerosol, vapour or mist of fine particles of the liquid or powder. This may be achieved by delivery of the aptamer using an applicator designed for this purpose. For example, the applicator may comprise a nebuliser, atomiser or vaporiser. In one embodiment, a drug delivery device may comprise a housing or container comprising the aptamer and an applicator which upon actuation forms the spray, aerosol, vapour or mist.

Aptamers according to the present invention may be conjugated to a cell delivery agent, such as a cell delivery peptide, e.g. see WO2009/147368. In one embodiment, the aptamer, pharmaceutical composition or medicament may be administered at a site of surgical resection. For example, where a subject has a cancer, some or all of the cancerous tissue at a given location in the subject's body may be surgically removed. After removal the aptamer, pharmaceutical composition or medicament may be applied to the remaining tissue. This may involve spraying the aptamer, pharmaceutical composition or medicament onto the tissue, or coating the tissue with the aptamer, pharmaceutical composition or medicament. Such application may be intended to kill remaining cancer cells, or inhibit their proliferation and thereby prevent reoccurrence or spread of the cancer. As such, in one aspect of the present invention a method is provided for reducing the risk of reoccurrence of cancer, the method comprising administering an aptamer,

pharmaceutical composition or medicament according to the present invention to a site of surgical resection of cancerous tissue. The administration may comprise spraying the aptamer, pharmaceutical composition or medicament onto tissue at the site of resection.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins. Eukaryotic translation initiation factor 4E (elF4e)

elF4e is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNA. It can exist in free form as a globular protein and as part of a multiprotein complex called elF4f. elF4e is essential for the translation of almost all cellular proteins.

The amino acid sequence of elF4e from Homo sapiens is available in Genbank under Accession no. NP_001124150.1 Gl:194578907. The elF4e protein may be from, or derived from, any animal or human, e.g. non-human animals, e.g. rabbit, guinea pig, rat, mouse or other rodent (including from any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate or other non-human vertebrate organism; and/or non-human mammalian animal; and/or human.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Brief Description of the Figures Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1. Selection of DNA aptamers against elF4e using a rapid selection procedure, a) A library composed of a looped 33-mer random sequence is amplified 10- fold by asymmetric PCR before negative selection with streptavidin beads and positive selection with elF4e beads followed by amplification with asymmetric PCR. b) Asymmetric PCR generates an abundance of single stranded aptamers and double stranded templates which is then removed by streptavidin beads, c) The number of unique sequences identified by high throughput sequencing decreases with increasing rounds of selection.

Figure 2. Aptamer clusters are genuine binders of elF4e a) Representative members of the top 12 clusters of aptamers selected, b) Relative abundance of each aptamer family as a percentage of all reads over increasing rounds of selection, (c) and d) 6% Polyacrylamide EMSA of the selected aptamers against elF4e and BSA at 2μΜ of aptamer and 150μΜ of protein (c) or stated concentration of protein (d). Aptamers are stained with SyBr Gold after electrophoresis.

Figure 3. elF4e aptamers inhibit translation, a-b) Insect cell transcription and translation kit using a luciferase template and stated dilution of aptamer (a) or 20μΜ of aptamer 1 in the presence of 120μΜ BSA or human elF4e (b). NS is an oligonucleotide that can form a hairpin structure c) Number of reads from elF4e Aptamer 1 cluster round 8 that deviated at specific positions from the most abundant member of the cluster, d) MFold prediction of the secondary structure of elF4e Apt 1. e) Insect cell transcription and translation assay with elF4e Apt 2 and eiF4e Apt 1 and mutants with a single modification at specific positions. All error bars reflect standard deviations. * p < 0.05; ** p < 0.01 ; *** p < 0.001 ; 2-tailed student's t test. For a), p-values are in comparison to control / no aptamer.

Figure 4. elF4e aptamers inhibit cell proliferation in HeLa and HEK293 cells HeLa and HEK293 cells were transfected with varying concentrations of aptamers using

Lipofectamine 2000 and cell viability measured with Cell Titer Blue for 3 hours 2 days after transfection. a) HEK293 cells transfected with 100nM of each aptamer. NS is an oligonucleotide that can form hairpin structures, (b) and c) HeLa and HEK cells transfected with varying amount of aptamers. ** p < 0.01 ; *** p < 0.001 ; 2-tailed student's t test compared to untreated cells.

Figure 5. Ligation of aptamers enhances activity, a) Aptamers phosphorylated with T4 DNA ligase and ligated at 4μΜ applied to the insect cell TnT kit and luciferase levels measured 4 hours after application. Aptamers heated and cooled were ligated, then heated to 85°C and cooled again instead of directly being applied to cells, b) HeLa cells were transfected with aptamers ligated or aptamers alone at 20nM with lipofectamine 2000 and measured 2 days after transfection with Cell Titer Blue.

Figure 6. Chart showing insect cell transcription and translation assay results using a luciferase template and 20pm of aptamer and incubated for 5 hours.

Figure 7. Table showing certain aptamer consensus sequences for each of clusters 1 to 12. Figure 8. Sequence of each aptamer identified by cluster.

Figure 9. Photographs showing fluorescence imaging of cells following spray application of aptamer in combination with Lipofectamine, PepFect14 (250pmoles or 1 nmoles) or with no vehicle.

Figure 10. Photographs showing fluorescence imaging of cells following spray application of peGFP-C1 plasmid in combination with Lipofectamine, PepFect14

(250pmoles or 1 nmoles) or with no vehicle. Figure 11. Photograph of spray bottles.

Figure 12. Table showing aptamer sequences for each of clusters 1 to 12, including consensus sequences. Figure 13. Charts showing insect cell transcription and translation assay results using a luciferase template and 20pm of aptamer and incubated for 5 hours. After incubation, luciferase activity was measured with 20% of reaction. After assay, 60% of reaction was diluted then RNA was Trizol extracted. Total RNA concentration was assayed by adding 1 x SyBr Gold to equivalent dilutions of the RNA and measuring fluorescence.

Figure 14. Chart showing rabbit reticulocyte transcription and translation assay results using a luciferase template and 20pm of aptamer and incubated for 5 hours. Figure 15. Chart showing results of HeLa cells transfection with 10OnM of aptamer using Lipofectamine 2000. Cell viability was measured 2 days after transfection with Cell Titer Blue. Figure 16. Photograph of gel electrophoresis. Aptamers were phosphorylated, heated and cooled before ligation with T4 DNA ligase overnight at 4 degrees Celsius.

Figure 17. Diagram of binding aptamer binding to elF4e on m7-GTP-beads, and photograph of electrophoresis. 150 nmoles of elF4e was incubated with 150 nmoles of each aptamer or water then incubated with m7-GTP-Sepharose beads containing ~500 nmoles of m7-GTP. Half the beads and half of the supernatant was then run on a polyacrylamide gel and Coomassie stained.

Figure 18. Photographs of gel electrophoresis. (A) Native TBE polyacrylamide gel with 5-fold or 10-fold molar excess of aptamer over elF4e. 10μΜ elF4e was incubated in PBS pH 8 with 5- to 10- fold excess of aptamers. elF4e is predicted to be slightly negatively charged at pH 8 and thus could migrate in the absence of SDS. Binding of aptamer to the protein is predicted to affect the migration of the native elF4e protein. (B) Native TBE polyacrylamide gel with 2μΜ Aptamer and 30μΜ of human elF4A1 or elF4B.

Figure 19. Charts and western blot showing that elF4e aptamers inhibit cell proliferation in HEK293 cells. (A) HEK293 cells transfected with shRNAs against elF4e or a control shRNA against DMPK TA cloned into pCR2.1. (B) HEK293 cells transfected with 100 nmol/l of each aptamer. (C) HEK293 cells were transfected with 100 nmol/l of biotinylated Apt 6 or biotinylated NS. Six hours after transfection, cells were fixed with paraformaldehyde and lysed. Cell lysates were then incubated with Streptavidin

Dynabeads. Western blots of the input, unbound, and bound fractions were probed with an elF4e antibody. **P <0.01 ; ***P < 0.001 ; two-tailed Student's i-test compared to untreated cells; n = 5.

Figure 20. MFold prediction structures for elF4e Apt 2, 6 and 7.

Figure 21. Chart showing cell viability of cells transfected with elF4e Aptamer 6 relative to untreated cells. B16F10 (murine melanoma), NSC34 (murine neuroblastoma), A549 (human lung adenocarcinoma), U20S (human osteosarcoma) and murine glioma cells were plated in 96 well plates in triplicate and transfected with 25nM, 50nM or 100nM of elF4e Aptamer 6. 72 hours later, cell viability was assayed using WST-1 proliferation kit, and viability was compared to viability of untransfected cells.

Examples Example 1 : Rapid identification and characterization of elF4e-inhibitinq DNA aptamer using high throughput sequencing

1.1 Abstract

Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High elF4e levels contribute to malignancies, thus elF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. The following example demonstrates a method for the rapid selection of looped DNA aptamers against elF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to elF4e and inhibit translation initiation in biochemical assays. When transfected into cells, elF4e aptamers cause a dramatic loss of cell viability in tumour cells, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, it is demonstrated that selection happens in waves, and that sequencing data can be used to infer aptamer structure. Lastly, it is shown that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest.

1.2 Introduction

Aptamers are single-stranded oligonucleotides that form secondary structures that can specifically interact with ligands (1 ,2), including small molecules (3), glycans (4) and proteins (5), for a wide range of functions including detection of its cognate target (6,7) and targeted delivery (8,9). Beyond just purely binding to their targets, DNA aptamers can also inhibit enzymatic activity and binding as well. Thrombin-binding aptamers have been shown to inhibit clot formation (5). AS1411 is another example of an inhibitory DNA aptamer. It binds to nucleolin and inhibits its interaction with arginine methyltransferase 5 (10). While a multitude of RNA aptamers and their analogs against various targets have been identified (REF), DNA aptamers are easier to perform a screen with as they are more resistant to nuclease digest and are amenable to standard PCR as opposed to the more tedious amplification processes for RNA and its analogs. The method of choice for identifying aptamers is called Systematic evolution of ligands by exponential enrichment (SELEX), in which specific and high affinity aptamers are identified through successful rounds of affinity selection and amplification with increasing stringency (typically 8-16 rounds). The initial aptamer library is frequently pre-cleared against the solid phase without the target protein to remove sequences that bind to the solid phase. For DNA aptamers, amplification with PCR after each round of selection is followed with a denaturation and purification step to isolate single stranded aptamers (11 ). Some groups have also used asymmetric PCR to preferentially generate aptamer strands over antisense complementary strands (12).

Following rounds of selection, the resultant library is typically then cloned and sequenced via Sanger sequencing. Recently, a number of groups (13,14,15,16,17) have harnessed high-throughput sequencing to the time-consuming cloning stage and dramatically increased the insight into the library sequence space that cannot be adequately covered with Sanger sequencing. While all these studies focused on the discovery of specific motifs, only two analyzed the increase in relative abundance of specific aptamers over time, which is more valuable than taking the end point analysis because some aptamers could dominate earlier on while other sequences appear much later in the selection rounds. Cho ei a! (13) analyzed the relative abundance of motifs over 3 rounds of selection while Schutze ei a/ (15) analyzed DNA aptamers selected to bind to streptavidin and traced the relative abundance of specific sequences through 10 rounds of selection. Interestingly, Schutze ei al (15) found two differing patterns of enrichment amongst the most abundant sequence clusters after 10 rounds of selection. The first group peaked in relative abundance between selection rounds 5-7 before declining in abundance to give way to the next wave of sequences between selection rounds 8-10. Another piece of data that could be gleaned from tracking the selection of specific aptamer clusters is the relative importance of specific substitutions, insertion or deletion in binding to the ligand. Although Schutze er a/ (15) and Ditzler et al (16) attempted to analyze the relative importance of particular positions to binding to its cognate target, analyses were complicated by the use of error-prone amplification processes inherent to reverse transcription and Taq polymerase-based PCR.

Cap-dependent translation in eukaryotes is initiated by the 5' end cap structure (m7GTP) of mRNA binding to elF4e. elF4e forms a complex with other initiation factors and the ribosomal 40S subunit, shuttling down the mRNA until the start codon is reached (18). Although elF4E regulates translation globally, overexpression of elF4e contributes to tumour malignancy by enabling the increased translation of mRNAs with highly structured, G+C-rich 5'UTRs, typical of proto-oncogenic mRNAs such as VEGF (19). Thus, elF4E inhibition can potentially be an anti— cancer strategy (20). Nucleic acid aptamers that can bind and inhibit elF4e activity can potentially be used to assay for expression of elF4e, screen for small molecule inhibitors that can mimic its action, even therapeutically to inhibit elF4e in a specific fashion. Although an RNA aptamer that binds to elF4e has been identified previously (21), RNA aptamers are long, thus costly to synthesize, and susceptible to nucleases, thus can degrade rapidly once applied, which limits the efficacy of the aptamers over time.

Here, a method is presented for the rapid selection of looped DNA aptamers against elF4e by combining negative selection and purification in a single step, followed by characterization of selection libraries with high throughput sequencing. The resulting aptamers show functional binding to elF4e and inhibit translation initiation in biochemical assays. When transfected into cells, elF4e aptamers can result in dramatic loss of cell viability, suggesting that these aptamers could be used for therapeutic ends. With the large data set provided by high throughput sequencing, it is shown that selection happens in waves and that sequencing data can be used to infer essential bases in aptamers. Lastly, it is shown that ligation of looped aptamers can enhance their biochemical effects in biochemical assays although not in cell culture experiments.

1.3 Materials and Methods

Protein expression, purification and biotinyiation. Rossetta pLysS competent bacteria were transformed with the pET11d expression plasmid containing the full-length elF4E clone. Both materials were kindly provided by p53lab A*STAR. The cells were grown in LB medium at 37°C to OD ₆ oo ~0.6 and elF4E induced with 1mM IPTG for 3 h at 37°C. Cells were resuspended in 50 mM Tris pH 8.0, 10 % sucrose, and sonicated. The sonicated sample was centrifuged for 10 min at 17,000 g at 4°C and resuspended in Tris Triton buffer (50 mM Tris pH 8.0, 2 mM EDTA, 100 mM NaCL, 0.5 % Triton X— 100). The sample was then centrifuged at 25,000 g for 15 min at 4°C and resuspended in Tris/Triton buffer. After re-centrifugation, the remaining pellet was solubilised in 6 M guanidinium hydrochloride, 50 mM Hepes-KOH pH 7.6, 5 mM DTT. The protein concentration of the sample was then adjusted to 1 mg/mL. The denatured protein was refolded via a 1/10 dilution into refolding buffer consisting of 20 mM Hepes-KOH, 100 mM KCI and 1 mM DTT. The refolded protein was concentrated and desalted using a

Amersham PD10 column into refolding buffer. The elF4E protein sample was run over a monoQ column and eluted with a 1 M KCI gradient. elF4E eluted as a sharp peak at a -0.3M KCI. elF4e protein was then biotinylated with EDC biotinylation kit (Pierce) and washed thrice with wash buffer with a 10KDa MW centrifugal ultrafiltration cartridge (centricon) to remove unbound biotin. Resultant protein (80pg) was conjugated to MyOne-Streptavidin beads (40μΙ) over 2 says at 4°C on shaker.

Aptamer Selection. The aptamer library (5 -

ACACTCTTTCCCTACACGACGCTCTTCCGATCT-tNJsa-AGATCGGAAGAGCTC-S was synthesized by Integrated DNA Technologies (Coralvi!le, Iowa, USA) and PAGE purified, exactly the same library as described in Hoon et al (17). Approximately 6 x 10 ¹² molecules (10 pmoles) out of a theoretical diversity of 7 x 10 ¹⁹ unique sequences was PCR amplified assymetrically with KOD polymerase (EMD4Biosciences), 100 pmoles of the L primer (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and 10 pmoles of the R primer (5'-[biotin]AGACGTGTGCTCTTCCGATCT-3') as per manufacturer's instructions to amplify each unique sequence ~10-fold. PCR annealing was performed at 57°C for 3 cycles (linear amplification of negative strand) then 65°C for 18 cycles (linear amplification of aptamer strand). Subsequent amplification of the aptamers retained by each round of selection by asymmetric PCR was performed with a similar protocol with 57°C for 20 cycles then 65°C for 15 cycles. The first 20 cycles is to allow for exponential amplification of the retained aptamer and the next 15 cycles for linear amplification of the positive aptamer strand. The resultant amplified library was then passed through 10μΙ of MyOne Streptavidin Dynabeads to remove double stranded PCR products and

Streptavidin-binding aptamers to produce single stranded non-streptavidin-binding DNA aptamers. 3μΙ of beads conjugated with each target protein was used for each aptamer selection thereafter, decreasing to 2μΙ and 1 μΙ for rounds 7 and 8 respectively. 25% of the prepared library from above was used for selection. 3 washes in PBST for 5 minutes was done in the first round, 4 washes in the 2nd, 5 washes in the 3rd, 7 washes in the 4th, 10 washes in the 5th and 12 washes in subsequent rounds. 50μΙ PCR off beads after selection using 1 μΙ 100μΜ L primer, 1 μΙ 10μΜ R primer (biotinylated), purified with streptavidin bead (1 ΟμΙ) to remove R primer (biotinylated) and 50% of previous round used for the 2nd round, 25% for the 3rd, 25% for the 4th round. 20% for the 5th round and 10% in subsequent rounds.

High Throughput Sequencing. Aptamer libraries were prepared by amplification of 1 μΙ of recovered and amplified selection with Primer GX1 (lllumina) and reverse primer with a unique barcoded sequence from ScriptSeq RNAseq barcode primers (Epicenter) using KOD polymerase. Barcoded libraries were pooled at roughly equimolar ratios based on band intensity on SyBr Gold stained polyacrylamide gel and the pooled sample was size selected by PAGE purification (6% TBE PAGE). The pooled size— selected sample was then sequenced on a Genome Analyzer llx for 36 cycles following manufacturer's protocols. The image analysis and base calling were done using lllumina's GA Pipeline. Adapters were trimmed with Biopieces remove_adapter script and remaining sequences were aligned and clustered according to sequence similarity.

Electrophoretic mobility shift assay. 20 pmoles of each aptamer was heated to 95°C and cooled in 10μΙ of PBS on ice followed by the addition of 0.5ul protein at the appropriate dilution. The mixture was then incubated for 5 minutes on ice before being loaded into a 10% polyacrylamide gel in Tris-borate buffer and subjected to

electrophoresis. The gel was then stained thereafter with 1x SyBr Gold (Life

Technologies) before visualization on a UV transilluminator.

Translation Assay. The assay was performed with TnT T7 Insect Cell Extract protein expression kit (Promega) and rabbit reticulocyte TnT protein expression kit (Promega). The kits generate capped mRNA with cap analogs and T7 RNA polymerase, before the insect or rabbit cell components translate the mRNA. 0.2μΙ of Luciferase ICE T7 Control DNA (provided in the kit) was mixed with 4μΙ of TnT T7 ICE Master Mix and 1 μΙ of aptamer / protein mix at the appropriate concentrations. The aptamer would have been heated to 95°C for 5 minutes in distilled water and cooled on ice immediately prior to addition. The reactions are mixed and incubated at 30°C for 5 hours and 2μΙ of sample is used for measurement of luciferase activity after addition of 20μΙ of Luciferase Assay Reagent (Promega) in a monochromator plate reader (Tecan Safire 2). To prepare ligated aptamers, aptamers were phosphorylated with T4 polynucleotide kinase (NEB) and heated to 95°C then cooled on ice before ligated using T4 DNA ligase at 10μΜ each.

Cell Culture and transfection. HEK293 and HeLa cells were cultured in DMEM High Glucose (Gibco, Life Technologies) supplemented with 10% FBS and antibiotics and incubated at 37°C in 5% C02. Transfection of cells were carried out with using

Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions, in 24 well plates.

Statistics. All experiments, unless otherwise stated, were all performed in triplicates. All error bars used in this report are standard deviations. Statistical significance was determined by one-tailed student's t-test assuming equal variance unless otherwise stated.

1.4 Results

Selection of elF4e binding aptamer

In order to maximize the chance of specific binding and for ease of selection, the biotin- streptavidin interaction was utilized for positive and negative selections as well as isolation of single stranded aptamers (Figure 1A), A 33-mer aptamer library previously utilized in Hoon et al (17) was synthesized with a loop design such that the 5' and 3' ends of the aptamer would form a loop. Approximately 6 x 10 ¹² unique sequences were amplified 10-fold with asymmetric PGR using a proof-reading DNA polymerase to increase the number of molecules representing each unique sequence. The biotinyiated reverse primer allows for the removal of double stranded DNA with streptavidin beads after PGR amplification (Figure 1 B) and also the concurrent removal of streptavidin- binding aptamers (negative selection). The heating and cooling steps within asymmetric PGR would also have allowed single stranded aptamer to form secondary structure, which means the unpurified flowthrough depleted of double stranded PGR products could be utilized for selection straight away against biotinyiated elF4e immobilized on streptavidin beads. Increasing the number of washes and decreasing the amount of protein-loaded beads and aptamers increased the stringency of selection with each round of selection. Each round of selection was amplified again with specific bar codes and sequenced. After removing poor quality sequencing reads, at least 2M reads per round was used for further analysis. The number of unique reads decreased with increasing round of selection (Figure 1 C), indicating that purifying selection is taking place.

Selection occurs in waves

After sequencing, the sequences from all rounds were grouped into clusters based on sequence similarity, with sequences differing by at most 3 bases grouped into the same clusters. The most highly represented members of 12 selected clusters without the loop sequences at either ends are shown in Figure 2A. Figure 2B shows the relative distribution of these clusters as a function of selection rounds. Based on the relative abundance, these aptamers can be broadly be binned into two classes. One class dominates early on in selection, peaking at rounds 3-6, examples of which are aptamers 1 , 3, 4, 7, 9, 10 and 12. The second class begins displacing the first class after round 7, examples of which are aptamers 2, 5, 6, 8 and 11. Schutze et al (15) also described two waves of selection, suggesting this is a generally applicable phenomenon with DNA aptamer selection. Hence, surveying aptamers at selection end points will invariably lead to omission of classes of aptamers, especially after 10-16 rounds of selection typical of SELEX. Surprisingly, there is no clear motif similarity between the 12 classes examined although several of the aptamer clusters are G-rich (aptamers 5, 6 and 8), suggesting that although G-quadruplex structures could have formed, most of the aptamers probably do not rely on G-quadruplexes for binding. In order to establish the aptamers could bind elF4e, electrophoretic mobility shift assay (EMSA) was performed on the 7 most highly represented aptamers against elF4e and BSA. These aptamers were shown to specifically bind to elF4e as opposed to BSA (Figure 2C). Three of the seven aptamers were shown to bind in a concentration-dependent manner and neither BSA nor Mdm2 (an untargeted protein) bound to the aptamers (Figure 2D). This demonstrates that the selection protocol yielded genuine binders.

Translation is inhibited by elF4e aptamers

As elF4e is a relatively small compact globular protein (25kDa), it is likely one of the aptamers would be bound to epitopes essential for its function, thus the aptamer could serve to inhibit elF4e function in cells. To establish if the aptamers could inhibit elF4e, we used an in vitro transcription and translation kit utilizing cap analogs, a luciferase expressing plasmid and T7 RNA polymerase to generate a mRNA mimic before the insect cell extract, with the translational machinery including elF4e, is used to convert the mRNA into protein. The relative luciferase activity would thus indicate translation initiation efficiency of elF4e in the presence of aptamers. Although all selected aptamers bound elF4e, only 3 out of the 7 inhibited translation at 20μΜ (Figure 6). This result is borne out in experiments in triplicate and Apt 1 , which showed dramatic inhibition of translation previously, demonstrated that this effect is dependent on the concentration of aptamer

(Figure 3A). To eliminate the possibility that elF4e aptamer affected transcription, the total RNA from the TnT reactions was DNase I treated then harvested by phenol— chloroform extraction to measure the total RNA produced by the T7-mediated transcription. The RNA content appears to be comparable, which demonstrates that luciferase inhibition was due to translation rather than transcriptional inhibition (Figure 13). To establish if the binding of Apt 1 to elF4e was responsible for translational inhibition, 25pg/pl of human elF4e added in the presence of Apt 1 compensated for the inhibition, suggesting that elF4e is indeed the target of Apt 1 -mediated inhibition (Figure 3B). Surprisingly, when the same experiment was performed using a rabbit reticulocyte transcription and translation kit, all aptamers surveyed except for Apt 5 and 7 resulted in inhibition of translation at 20 μΜ (Figure 14), suggesting that the binding surfaces of the aptamers are different, hence aptamer 1 and 6 are capable of inhibiting both insect elF4e and rabbit elF4e but aptamers 2-4 are only capable of affecting mammalian elF4e. Thus, it was successfully demonstrated that the selected aptamers bound to elF4e to inhibit translation in a biochemical system.

Sequence determinants of elF4e inhibition can be deduced from sequence abundance

As high throughput sequencing was used to survey the library space, a large dataset of sequences could be probed. Furthermore, the use of proof-reading polymerases for amplification of the libraries reduced the chance of random mutations being responsible for sequence variation within a cluster. Thus, it was hypothesized that the relative abundance of specific sequence substitutions in each position of the consensus could be used to predict how essential the base is to its function. Sequences from cluster 1 was curated and all unique sequences with at least 3 reads per million in the final round of selection were used to construct the relative abundance of each substitution (Figure 3C). Based on this data, mutations made in the red positions are predicted to negatively impact its binding and hence inhibition because they are poorly represented. Highly represented mutations in green, is conversely predicted to not affect its function. The Apt 1 secondary structures predicted by MFold22 to have the lowest free energy and the relative positions of the mutated residues are shown in Figure 3D. As predicted, changing positions 7, 11 and 21 all resulted in a significant increase in luciferase activity which is indicative of reduced binding, while changing position 22 does not seem to result in a big difference in elF4e mediated inhibition (Figure 3E). The only unexpected result was a substitution at position 29, which did not seem to be affect elF4e inhibition, despite having no aptamers deviate from the consensus. This result demonstrates the first example of sequencing data being used to infer essential residues for function and hints at the possibility of designing further mutagenesis of non-essential residues to improve function. elF4e aptamer result in dramatic loss of cell viability in HeLa and HEK293 cells Since elF4e knockdown reduces cancer cell proliferation (23), it is possible that elF4e inhibition with aptamers can affect cell proliferation. Thus, 7 aptamers were transfected at 100nM with lipofectamine in HEK293 cells and viability measured after 2 days using a redox-based cell viability assay (Figure 4A). As controls, a hairpin-forming single stranded DNA was used (NS seq). Aptamer 6 showed the strongest inhibition, dramatically reducing cell viability to approximately 3% of negative controls. Furthermore, this inhibition is concentration-dependent (Figure 4B) and is also effective in HeLa cells (Figure 4C and Figure 15).

Ligation enhances inhibitory efficacy of elF4e aptamers

Bifunctional aptamers, which contain two aptamers targeted to different epitopes on thrombin, have been shown to enhance the activity of inhibitory aptamers (24).

Concatamerization of linear aptamers also appear to increase the affinity of HER2 DNA aptamers to HER2 (25). As the elF4e aptamers were designed as loops with blunt ends, they are amenable to ligation. Phosphorylation of aptamers does not seem to alter its ability to inhibit translation at 4μΜ concentration (Figure 5A vs Figure 3A). Subsequent ligation of these aptamers resulted in either homodimerization or heterodimerization as demonstrated by a shift in migration in a polyacrylamide gel (Figure 16) and both types of dimerization increased the efficacy of the aptamers at inhibiting translation and the effect is not affected by denaturation and refolding of the ligated circular aptamer (Figure 5A). Surprisingly, the same effect could not be seen with elF4e inhibition in vitro, which in fact seemed to reduce the efficacy of the aptamers (Figure 5B).

1.5 Discussion

DNA aptamers offer an alternative to antibodies in the therapeutic and diagnostic space, with key advantages of increased stability and ease of synthesis and modification. This is counterbalanced by the lack of chemical diversity on the backbone of the aptamers, which limits the types of interactions possible with ligands and consequently results in lower affinity as compared to antibodies. However, the development of modified (25) or unnatural (26) bases in aptamer libraries has partially ameliorated the problem and it is likely that aptamers can make up for weaker contact interactions with favourable secondary structures that fit better. One of the key challenges towards greater adoption of aptamer technology is the development of a rapid selection protocol to build a database of aptamers against diverse ligands to facilitate technology uptake. To this end, a DNA selection protocol has been developed which does not require extensive cleanup between selections.

The other limiting factor, prohibitively high costs of characterizing libraries obtained from a screen, has recently been solved with the graduation from Sanger sequencing to high throughput sequencing with the associated huge dataset. As discussed in the

introduction, several groups have already managed to utilize high throughput data to identify sequence motifs for binding. However, no study has thus far used deviation from a consensus as a mapping tool for essential bases because few studies utilize proofreading enzymes in library amplification, which confounds inference that can be drawn from deviation from the consensus as that could be just a PCR error. Another inference that may be drawn from the high throughput sequencing dataset is the binding characteristics of the aptamer to its target. It was noted that selection happened in waves, with the first wave dominating when the number of washes was small and the second wave when the number of washes increased. Based on this, it may be possible to classify the first group of aptamers as having low dissociation constants and strong binding and the second waves as having low off-rates and weaker binding. Future kinetic studies may be able to substantiate this hypothesis and shed light on the nature of the sequences.

Knowing the relative dissociation constants and off-rates can aid in designing conjugates for targeting or inhibition. The aptamers discovered are also interesting because there is no sequence or structural similarity to an elF4e-binding inhibitory RNA aptamer (21 ). Moreover, no obvious consensus motif could be obtained from alignment of all clusters. The diversity of secondary structures of the clusters predicted by MFold (Figure 20) also suggests that the clusters obtained were not binding to elF4e in the same fashion, suggesting different epitopes. Furthermore, ligation of aptamer clusters seem to enhance their activity in a test tube, suggesting that they bound to different epitopes and worked in a co-operative fashion to increase binding of the target. Thus, it may be generally applicable to identify multiple aptamers targeting different epitopes, each with moderate dissociation constant, and construct multimers that possess the low dissociation constant that will fulfil clinical needs.

Here, a selection protocol is presented that has the following advantages. It is rapid, requiring only two steps between rounds of selection to regenerate and produce functional single stranded DNA aptamers while performing negative selection

simultaneously. It also uses high throughput sequencing data to characterize evolution of aptamer populations while producing data that predicts functional importance of specific bases in each aptamer cluster. Due to the design, aptamer sequences selected are amenable to conjugation via ligation, which produces multifunctional aptamers that are functionally better than the monomers. This protocol is thus capable of enhancing the odds of identifying functional specific aptamers. Together with other rapid screening protocols for aptamers, this protocol will hopefully increase the uptake and utilization of aptamer-based reagents by simplifying aptamer selection.

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2. Tuerk, C, Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science,249,505-10.

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6. Stojanovic.M.N., de Prada.P., Landry.D.W. (2001 ) Aptamer-based folding fluorescent sensor for cocaine. J Am Chem Soc, 123,4928-31. 7. Gronewold.T.M., Glass.S., Quandt.E., Famulok.M. (2005) Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors. Biosens Bioelectron. ,20,2044-52.

8. McNamara,J.0.2nd, Andrechek.E.R., Wang.Y., Viles.K.D., Rempel.R.E., Gilboa.E., Sullenger.B.A., Giangrande.P.H. (2006) Cell type-specific delivery of siRNAs with aptamersiRNA chimeras. Nat Biotechnol,24, 1005-15.

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13. Cho.M., Xiao.Y., Nie.J., Stewart.R., Csordas.A.T., Oh.S.S., Thomson.J.A., Soh.H.T. (2010) Quantitative selection of DNA aptamers through microfluidic selection and high- throughput sequencing. Proc Natl Acad Sci U S A., 107,15373-8.

14. Kupakuwana.G.V., Crill.J.E. 2nd, McPike.M.P., Borer.P.N. (2011 ) Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing. PLoS One. ,6(5),e19395.

15. Schiitze.T., Wilhelm.B., Greiner,N., Braun.H., Peter.F., Morl.M., ErdmannNA, Lehrach.H., Konthur.Z., Menger.M. et al. (2011 ) Probing the SELEX process with next- generation sequencing. PLoS One,6,e29604. 16. Ditzler.MA, Lange.M.J., Bose.D., Bottoms.C.A., Virkler.K.F., Sawyer.A-W.,

Whatley,A S., Spollen,W., Givan,S.A., Burke,D.H. (2013) High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase. Nucleic Acids Res.,41 , 1873-84. 17. Hoon.S., Zhou.B-. Janda.K.D., Brenner.S., Scolnick,J. (2011 ) Aptamer selection by highthroughput sequencing and informatic analysis. Biotechniques,51 ,413-6.

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Example 2: Spray application of aptamers

HEK293 cells were plated at 70,000 cells / well in a 24-well plate. One day after plating, 30pmoles / 500ng of fluorescein labelled Mdm2-binding aptamer (single stranded oligonucleotide 53 nt in length) or 500ng of peGFP-C1 (a DNA plasmid encoding EGFP- C1 under control of CMV promoter) were complexed with 1 μΙ of lipofectamine 2000 as per manufacturer's instruction or with 250pmoles / I nmole of PepFect14 as described in Ezzat et al (PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Res. 2011 Jul;39(12):5284-98. doi:

10.1093/nar/gkr072. Epub 2011 Feb 23) or left uncomplexed. The complexes were then added to 100μΙ OptiMEM.

The complexes in OptiMEM were either added to 400μΙ of medium in the well, or applied onto cells without medium by directly adding by pipetting or spraying on with a 5ml spray bottle (Figure 11 ) (3 sprays is ~ 100μΙ). 10 or 30 minutes after delivery, 400μΙ of medium is added.

One day after, cells were imaged for green fluorescence. Figures 9 and 10 demonstrate that spraying on the complexes was as effective a method of delivery as direct addition to cells. Notably, spraying of naked DNA especially the single stranded oligonucleotide, but not addition of naked DNA by pipette, results in significant fluorescence in the cells.

Example 3: Analysis cell viability of cells transfected with elF4e Aptamer 6 relative to untreated cells.

B16F10 (murine melanoma), NSC34 (murine neuroblastoma), A549 (human lung adenocarcinoma), U20S (human osteosarcoma) and murine glioma cells were plated in 96 well plates in triplicate, transfected with 25nM, 50nM or 100nM of elF4e Aptamer 6 with 2μΙ of lipofectamine 2000 per of oligonucleotide, in 100μΙ of medium. 72 hours later, the WST-1 proliferation kit was used to assay for cell viability. The measured viability was then compared to the viability of untransfected cells.

Figure 21 shows that Glioma, A549 and U20S cells were all responsive to elF4e- mediated inhibition of proliferation, albeit to different extents. B16F10 and NSC34 cells were less susceptible to elF4e-mediated inhibition, with marginal activity seen at 100nM with B16F10, and no activity at all with NSC34 cells (Figure 21 ).