Optimization method of a polynucleotide sequence

Abstract

The present invention provides for a method of optimisation of a polynucleotide sequence for use in an aptamer based assay, comprising adding additional bases to the ligand binding domain. The invention also covers methods of detecting target molecules in a sample using 5 optimised polynucleotide sequences in a suitable detection assay.

Claims

1. A method for selecting an aptamer for use in a particle detection assay, the method comprising: (i) providing a plurality of aptamers, wherein each aptamer comprises: (a) a ligand binding domain selective for a target substrate; and (b) at least one non-ligand binding domain comprising one or more nucleotides, wherein the at least one non-ligand binding domain promotes a direct association between the aptamer and a particle; and wherein each aptamer differs only in the number and sequence composition of the non-ligand binding domain nucleotides; (ii) associating each aptamer with a particle to form a non-covalent aptamer-particle complex in the absence of the target substrate; (iii) incubating the aptamer-particle complex with a target substrate in the presence of salt, wherein the aptamer dissociates from the particle when binding to the target substrate; (iv) measuring a signal transduction produced in step (iii) in the particle detection assay; and (v) selecting an aptamer which generates an optimized signal transduction measured in step (iv) when compared to the signal transduction generated by the other aptamers from the plurality of aptamers tested.

2. The method according to claim 1, wherein the non-ligand binding domain is located at the 5 end, the 3 end, or both the 5 and 3 ends of the ligand binding domain.

3. The method according to claim 1, wherein the non-ligand binding domain comprises 1 to 10 nucleotides.

4. The method according to claim 1, wherein the aptamer is an ssDNA or an RNA molecule.

5. The method according to claim 1, wherein the particle is a nanoparticle, microparticle, or quantum dot.

6. The method according to claim 1, wherein the assay is a colorimetric assay.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 (A) is a schematic depiction of aggregation in the colorimetric assay of E2; (B) Shows saturation binding curve of E2 with the NP-35-mer, NP-22-mer and NP-75-mer aptamers. Experimental data (from plotting normalised unbound fraction as 1/fa against 1/E2 concentration) was fitted using a linear regression function.

(2) FIG. 2 shows determination of the optimal salt concentration (indicated by the dashed arrow) for signal generation by comparison between the salt dependent aggregation of AuNPs (.square-solid.) AuNP-75-mer aptamer (.circle-solid.), and AuNP-75-mer aptamer in the presence of 100 nM E2 (.box-tangle-solidup.). Aggregation is measured via the relative absorption at 523 nm as indicated in the inset.

(3) FIG. 3 shows raw UV-vis spectra for sensing E2 using the 75-mer aptamer, 0 nM (), 5 nM (), 100 nM (), 200 nM () and 400 nM ().

(4) FIG. 4 shows colorimetric response towards a range of E2 concentrations for the AuNP-75-mer aptamer () when compared with bare AuNPs () and AuNP-poly-T controls (). The colour change over the range is from pink/red colloidal solution to purple/blue. Error bars indicate standard deviation of the mean of three experiments.

(5) FIG. 5 shows specificity examination of the 75-mer aptamer towards a number of interfering targets at 200 nM concentration. Error bars indicate standard deviation of the mean of three experiments.

(6) FIG. 6 Shows secondary structure of the 75-mer aptamer predicted by M-fold program indicating the truncation positions. The sequence shown in this Figure corresponds to SEQ ID NO: 1.

(7) FIG. 7 shows CD spectra for 400 nM of the 75-mer incubated with 0 M (.circle-solid.) E2 and 10 M E2 (.box-tangle-solidup.).

(8) FIG. 8 shows CD spectra for 600 nM of the 35-mer aptamers, incubated with 0 M E2 (.circle-solid.) and 10 M E2 (.box-tangle-solidup.).

(9) FIG. 9 shows determination of the optimal salt concentration, indicated by the dashed arrow at 24 mM, for signal generation comparison between the salt dependent aggregation of AuNPs (.circle-solid.), AuNP-35-mer aptamer (.diamond-solid.), and AuNP-35-mer aptamer in the presence of 100 nM E2 (.box-tangle-solidup.).

(10) FIG. 10 shows colorimetric responses towards a range of E2 concentrations using the AuNP-35-mer aptamer () when compared with bare AuNPs () and AuNP-poly-T () controls (figure inset indicated an extended linear response of the same figure). Error bars indicate standard deviation of the mean of three experiments.

(11) FIG. 11 (A) shows the calculated binding domain for the 22-mer E2 aptamer; (B) shows the data set for the 22-mer aptamer system compared with the bare NP and a random 22 aptamer the non-random 22-mer system delivers detection limits of around 200 pM. However, the magnitude of absorption change is reduced compared to the 35-mer aptamer. The sequence shown in this Figure corresponds to SEQ ID NO: 1.

(12) FIG. 12 Shows the selectivity of the AuNP-35-mer aptamer colloidal solution for target small molecules at 800 pm concentration. Error bars indicate standard deviation of the mean of three experiments.

(13) FIG. 13 TEM image (left) and photographs (right) showing the pinkish colour of AuNPs+0.1 nmol 75-mer aptamer

(14) FIG. 14 TEM image (left) and photographs (right) showing the pinkish-red colour of AuNPs+0.1 nmol 75-mer aptamer and the detection of 100 nM E2.

(15) FIG. 15 TEM image (left) and photographs (right) showing the purplish-blue colour of AuNPs+0.1 nmol 75-mer aptamer and the detection of 400 nM E2.

(16) FIG. 16 shows DLS size characterisation for AuNPs+0.1 nmol 75-mer aptamer (.square-solid.) and the detection of 100 nM E2 (.circle-solid.) and 400 nM (.box-tangle-solidup.).

(17) FIG. 17 (A) Salt dependent aggregation of AuNP-35-mer aptamer and AuNP-35-mer aptamer+100 nM E2 in rat urine (optimal salt concentration indicated by the black arrow). Photographs of AuNPs and AuNP-35-mer aptamer before and after addition of 5 L rat urine are shown in the inset, legend: .circle-solid.=35-mer+100 nM E2, .square-solid.=35-mer; (B) calorimetric aptasensor response towards a range of E2 concentrations in spiked rat urine using the AuNP-35-mer and AuNP-75-mer aptamers (photograph of the same samples in the top panel) compared with AuNP-poly-T control, legend: -35-mer, -polyT, -75-mer. (C) Specificity examinations of interfering molecules (at 200 nM) in rat urine samples using the AuNP-35-mer aptamer colloidal solution. Error bars indicate standard deviation of the mean of three experiments.

(18) FIG. 18 (A) Salt dependent aggregation of AuNP-75-mer/AuNP-75-mer random DNA and AuNP-75-mer aptamer/AuNP-75-mer random DNA+100 nM E2 (optimal salt concentration indicated by the black arrow); (B) Salt dependent aggregation of AuNP-35-mer/AuNP-35-mer random DNA and AuNP-35-mer aptamer/AuNP-35-mer random DNA+100 nM E2 (optimal salt concentration indicated by the black arrow); (C) Salt dependent aggregation of AuNP-22-mer/AuNP-22-mer random DNA and AuNP-22-mer aptamer/AuNP-22-mer random DNA+100 nM E2 (optimal salt concentration indicated by the black arrow); (D) Same as (B) but in urine samples.

(19) FIG. 19 shows Standard calibration curve of the conductivity of NaCl solutions to estimate the ionic strength of rat urine. 10 L of the rat urine was diluted to 15 mL, using Milli-Q water, and the conductivity was measured. The final ionic strength of the rat urine sample is 2.1 mM; legend: - - - diluted rat's urine, conductivity calibration curve using NaCl.

(20) FIG. 20 shows a photograph (top) and UV-vis spectra of the specificity results using the 35-mer aptamer in rat urine samples spiked with 200 nM BPA, BPF, progesterone (P4) and testosterone (T), and E2; legend: .circle-solid.-BPA, .square-solid.-control, -E2, .box-tangle-solidup.-BPF, .Math.-T, .diamond-solid.-P4.

(21) FIG. 21 shows the absorption spectra of E2 sensing in rat urine samples using the 75-mer aptamer; legend: .square-solid.-0 E2, .circle-solid.-5 M E2, .box-tangle-solidup.-15 M E2, .Math.-25 M E2, .diamond-solid.-35 M E2, -50 M E2.

(22) FIG. 22 shows determination of the dissociation constant (K.sub.D) for the 35-mer aptamer. a) Representative emission spectra of E2 in BWB. b) Calibration curve of E2 and fluorescent intestines of E2 after reaction with NP-35-mer. c) Saturation binding curve of E2 with the NP-35-mer. Experimental data (from plotting normalised unbound fraction f.sub.a against E2 concentration) was fitted using a non-linear regression function as explained in the Methods section in the main text.

(23) FIG. 23 shows a colorimetric response of aptamers selective for BPA. Error bars indicate standard deviation of the mean of three experiments. Legend: Random 75-mer DNA; : 75-mer BRA aptamer; : 42-mer BPA aptamer.

(24) FIG. 24 shows the 2D structured of the 75-mer BPA aptamer. indicates estimated start and end points comprising central loop points of the ligand binding; indicates the start and end points of the 42-mer BPA aptamer. The sequence shown in this Figure corresponds to SEQ ID NO: 1.

EXAMPLES

(25) 17-estradiol (E2), progesterone, testosterone, Bis(4-hydroxyphenyl methane) (BPF), bisphenol-A (BRA), E2 75-mer aptamer, truncated version 35-mer, poly-thymine (18) (poly T) Chloroauricacid (HAuCl.sub.4) and sodium chloride (NaCl) are purchased from Sigma-Aldrich. The aptamers are dissolved in Milli-Q water and stored at 5 C. prior to use. Milli-Q water is used in all experiments (unless stated), and all other chemicals are of analytical grades purchased from standard chemical suppliers.

(26) The aptamers of the present invention can be synthesised by standard synthetic methodologies commonly known and understood by those in the art for example, synthesis by SELEX.

(27) General Procedure for the Synthesis of NPs:

(28) Nanoparticles suitable for application with this invention can be prepared according to standard literature methods. For example: synthesis of AuNPs is described in Jana et al., 2001; synthesis of PtNPs is described in Teranishi at al., 1999; synthesis of AgNPs, is described by Yin at al., 2002; synthesis of PdNPs is described by Ge et al., 2007; synthesis of CoNPs is described by Redel et al., 2009; and synthesis of CuNPs can be found in Wu and Chen, 2004.

(29) Procedure for Synthesis of AuNPs:

(30) HAuCl.sub.4 (100 mL, 1 mM) is reduced with sodium citrate (10 mL, 38.8 mM) to provide AuNPs of 10 nm diameter (FIG. 13). [Grabar, K. C., at al, Anal. Chem., 1995, 67, 1217-1225]. An aqueous solution of HAuCl.sub.4 (100 mL, 1 mM) is vigorously stirred at 250 C. for 10 min and a solution of sodium citrate (10 mL, 38.8 mM) is added at once. The solution is boiled for 10 minutes, and is stirred for another 15 minutes at room temperature. AuNPs solution is stored at 4 C. prior to use. The concentration of AuNPs is calculated according to the Beer-Lambert law, using an extinction coefficient of 2.7 10.sup.8 M.sup.1 cm.sup.1 at 525 nm [Haiss, W., et al., Anal. Chem., 2007, 79, 4215-21]. The concentration of the AuNPs is estimated to be 14 nM.

(31) General Procedure for AuNP-Aptamer Blending and Coincubation:

(32) Those of skill in the art will realise that the procedure for coating the NPs is a standard general procedure and can be applied to other NPs suitable for application with the invention. AuNPs are used for exemplification and the procedure is not restricted only to AuNPs.

(33) AuNPs are purified by a 1:10 dilution of AuNPs in Milli-Q water, centrifugation at 12,500 rpm for 15 minutes (MIKRO 120-Hettich). The AuNPs are then re-suspended in Milli-Q water at the original 1:10 dilution. The removal of the excess citrate is confirmed by -potential values of (33.46 mV (0.35)) before and (23.5 mV (0.28)) after purification. A 0.1 nmole solution of the aptamer in Milli-Q water, or poly-T in the case of control experiments, is prepared and immediately added to the purified AuNPs suspended in 0.3 mL of Milli-Q water, to provide an aptamer concentration of 33.3 nM for the E2 aptamers (SEQ ID Nos: 1, 2, 3, 4, 5, 6), and 100 nM for the BPA aptamers (SEQ ID Nos: 7 and 8), with a aptamer: particle number ratios of 3:1 and 9:1, respectively, for a particle number of 2.510.sup.13. The NP-aptamer samples are prepared 1 hour prior to sensing experiments.

(34) General Procedure for Salt Titration Experiments:

(35) Those of skill in the art will realise that this procedure is a standard general procedure and can be applied to other NPs suitable for application with the invention. AuNPs are used for exemplification and the procedure is not restricted only to AuNPs.

(36) The ionic strength of the aptamer solution is independently adjusted to the values stated in FIG. 9, by adding different volumes of 0.5 M NaCl to 100 L samples of bare NPs, NP-aptamer, or NPs-aptamer+100 nM E2 or BPA. For example, the optimum ionic strength for the AuNP-75-mer E2 (SEQ ID No:1) and the AuNP-75-mer BPA (SEQ ID No:7) aptamer system is 23.8 mM. The optimum ionic strength for the AuNP-42-mer BPA aptamer (SEQ ID No:8) system is 14.5 mM The samples are allowed to stand for 15 minutes before measuring the degree of aggregation by UV-vis absorption at 523 nm.

(37) General Procedure for K.sub.D Measurement:

(38) The procedure for estimating the K.sub.D of the 35-mer was based on that used for the 75-mer [Alsager, O. A; Kumar, S.; Willmott, G. R.; McNatty, K. P.; Hodgkiss, J. M. Biosens. Bioelectron. 2014, 57C, 262]. The aptamer was immobilized on polystyrene nanoparticles, exposed to aliquots of E2, and the fraction bound E2 was measured via UV fluorescence and fit to a binding isotherm after separation of the nanoparticles from the supernatant. 200 nmol of EDC and NHS (20 L of 0.01 M in MES) was added to activate 400 L of carboxylate polystyrene nanoparticles (NPs, 5.210.sup.10 particle mL.sup.1) in MES for 40 min, followed by addition of 0.1 nmol of 35-mer aptamer and incubation overnight. The samples were centrifuged at 14000 rpm for 30 min and the supernatant was discharged. 1 mL E2 with various concentrations in BWB containing 5% ethanol was added to the samples, sonicated for 10 mins, and incubated overnight. The samples were then centrifuged at 14000 rpm for 30 min, the supernatant was isolated, transferred to a 1 cm quartz cuvette, and the fluorescence of the unbound E2 was collected via 279 nm with a Shimadzu RF-5301PC spectrofluorophotometer. The measured fluorescence intensities at 310 nm were first converted to concentration via an E2 calibration curve. By expressing a measured E2 concentration as an unbound fraction, f.sub.a, and plotting against total E2 concentration, the K.sub.D was determined as 10 nM by fitting to the binding isotherm in equation 1. We observed a saturation offset at f.sub.a=0.6 due to partial loss of the NP-35-aptamer conjugate during the centrifugation, and renormalized to f.sub.a (max)=1.

(39) $\begin{array}{cc}{f}_a=\frac{\left[E2\right]}{K_D+\left[E2\right]}& \left(1\right)\end{array}$

(40) Procedure for E2 Detection:

(41) Those of skill in the art will realise that this procedure is a standard general procedure and can be applied to other NPs suitable for application with the invention. AuNPs are used for exemplification and the procedure is not intended to be restricted only to AuNPs.

(42) Sample water is collected from the Hutt River, Wellington, New Zealand and pre-treated by stirring 50 mL overnight at room temperature with 1 g of activated charcoal and filtering twice through 0.22 m syringe-filters to provide treated water. The conductivity of the treated water is measured as 100 s cm.sup.1 (at 25 C. with the pH is 8) and indicates there is a very low salt concentration in the sample. Alternatively, Milli-Q water is used instead of treated river water.

(43) Stock solutions of the target small molecules and/or other target substrate are made in ethanol before adding appropriate volumes to the treated water or Milli-Q water, and adjusting the final ethanol content to 5%, ensuring sufficient target small molecule and/or other target substrate solubility. 20 L of the pre-treated test samples are added to 100 L of AuNP-aptamer solution to obtain varying E2 concentrations and provide a total reaction volume of 120 L.

(44) Control samples are made up from blank water containing 5% ethanol. Samples are incubated for 10 minutes at room temperature to facilitate binding to the target. The optimised NaCl concentration determined from the salt titration experiments is added to the target detection solutions (10 mM for bare NPs, 23.8 mM for poly-T/E2 aptamer samples), followed by gentle shaking, The samples are visually inspected after 15 minutes, and the UV-vis absorption of 5 L aliquots is measured using a Thermo Scientific NanoDrop 1000 Spectrophotometer.

(45) Procedure for BPA Detection:

(46) The same procedure outlined above for E2 detection was followed, but with the target substituted for BPA, and the aptamers substituted for BRA aptamers.

(47) Animal Urine Study:

(48) Rat urine is collected from sexually mature ship rats (Rattus rattus), filtered with 0.22 m syringe-filters, and spiked with E2 and interfering molecules after adjusting the content of ethanol to 5% (control rat urine sample comprised blank rat urine containing 5% ethanol). 5 L of spiked urine is added to 100 L AuNP-poly-T, AuNP-75-mer aptamer or AuNP-35-mer aptamer, incubated at 50 C. for 10 min, followed by addition of optimised NaCl (57.4 mM), gentle shaking, visual inspection after 15 min and measurement of UV-vis absorption as described above.

(49) -Potential Measurements for Au Nanoparticles:

(50) Those of skill in the art will realise that this procedure is a standard general procedure and can be applied to other NPs suitable for application with the invention. AuNPs are used for exemplification and the procedure is not restricted only to AuNPs.

(51) 120 L samples of bare AuNPs, AuNP-aptamer, and AuNP-aptamer in the presence of 100 nM E2 in Milli-Q water, are incubated at room temperature for 1 hour and are centrifuged at 12,500 rpm for 15 minutes. The excess aptamer is removed by decantation of the supernatant and the NPs are re-suspended in 1 mL Milli-Q water. Samples are loaded in a folded capillary cell, inserted into a Zetasizer Nano ZS equipped with a 633 nm laser (Malvern Instruments, UK) and equilibrated at 25 C. for 2 minutes prior to measurement. Measurements are made in triplicate, with fixed parameters of pH 7, viscosity 0.887 mPa s, and refractive index of 1.33. The measurements are reported as average valuestandard deviation (Table 2).

(52) TABLE-US-00002 TABLE 2 -Potential Values and Surface Densities for Different Samples Investigated during E2 Sensing -potential.sup.a a surface density.sup.b sample No E2 1 M E2 No E2 20 M E2 bare AuNPs 23.5 (0.3) 24.0 (0.7) 75-mer aptamer 40.2 (0.9) 32.3 (0.9) 1.12 0.48 75-mer random 46.2 (0.7) 47 (1) 1.01 1.04 35-mer aptamer 29 (1) 25.0 (0.2) 4.42 1.3 35-mer random 41 (1) 41.7 (0.3) 4.08 3.4 22-mer aptamer 29 (1) 24.3 (0.8) 4.2 2.1 22-mer random 42 (2) 38.0 (0.7) 4.9 4.6 .sup.amV (STD, n = 3). .sup.b/10.sup.13 molecule/cm.sup.2.

(53) CD Studies:

(54) 1 mL solutions of the 75-mer and 35-mer and 22-mer aptamers at 400 nM, 600 nM and 600 nM, respectively, are prepared in water containing 5% ethanol, 23.8 mM NaCl, and 0 or 10 M of E2. Samples are measured in a 1 cm path length quartz cell. CD spectra are measured using a Chirascan CD spectrometer instrument over the wavelength range from 200 to 400 nm, scanned at 200 nm per minute.

INDUSTRIAL APPLICATION

(55) The present invention provides useful methodology for the detection of target substrates in samples. The present invention may find use is the field of environmental, forensic, diagnostic testing of samples.

REFERENCES

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