PNA probe

Abstract

Disclosed herein are improved PNA based monomers, nucleobases, oligomers and probes for use in a variety of different methods of analysing nucleic acids. Further, the disclosure provides methods of preparing the modified and improved PNA molecules as well as methods of using the same.

Claims

1. A PNA oligomer, wherein the PNA oligomer has the general formula: ##STR00030## wherein: G is a charged moiety, or a moiety capable of carrying a charge at a pH in the range of 6-8; NB is a nucleobase; and l≥1; m≥1; and n≥0.

2. The PNA oligomer of claim 1, wherein the total number of PNA units (l+m+n) in the oligomer is in the range of 12-24.

3. The PNA oligomer of claim 1, wherein n+m is in the range of 3-5.

4. The PNA oligomer of claim 1 wherein the ratio of the number of units having a “G” moiety in formula (V), to the total number of repeat units, is in the range of 1:10-1:2.

5. The PNA oligomer of claim 1, wherein m=1.

6. The PNA oligomer of claim 1, wherein the PNA molecule is covalently attached to a solid support.

7. The PNA oligomer of claim 1, wherein n≥1.

8. A method for preparing a PNA oligomer according to claim 1, the method comprising reacting one or more PNA monomers so as to form the PNA oligomer, wherein the one or more PNA monomers comprise at its gamma position a charged moiety or a moiety capable of carrying a charge at a predetermined pH, and wherein the one or more PNA monomers each have the general formula: ##STR00031## wherein: G is a charged moiety, or a moiety capable of carrying a charge at a pH in the range of 6-8; P.sub.1 is a protective group P, or is hydrogen; P.sub.2 is a protective group P, or is hydrogen, or is a group selected from the list consisting of alkyl, cycloalkyl, aryl, aralkyl, or halogen, P.sub.3 is hydrogen, or is a protective group P, or is a group represented by formula (II) below: ##STR00032## wherein NB is a nucleobase.

9. A method according to claim 8, the method comprising the preliminary step of covalent bonding a/the PNA monomer on a solid support through its C-terminal.

10. A method of characterising a nucleotide in a nucleic acid sequence, said method comprising the steps of: ##STR00033## wherein Y is a functional group capable of reversible covalent reactions; X.sub.1-X.sub.4 is a detectable tag, spacer-tag combination or hydrogen; and Z is carbon or nitrogen; wherein the PNA oligomer comprises a moiety capable of reacting reversibly with functional group Y and wherein the modified base which integrates with the nucleic acid/PNA duplex is complementary to that of the nucleotide to be characterised, the nucleotide being characterised by mass spectrometry or by means of the detectable tag of the modified base.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The present invention will now be described in detail and with reference to the following Figures which show:

(2) FIG. 1: Reactions schemes used to prepare a PNA monomer according to an embodiment of the invention;

(3) FIG. 2: Scheme representing a colorimetric approach to test PNA probes;

(4) FIG. 3: Examples of PNA molecules tested;

(5) FIG. 4: Results of the intensity measured for the PNA molecules of FIG. 3;

(6) FIG. 5: Alternative examples of PNA molecules tested;

(7) FIG. 6: Results of the intensity measured for the PNA molecules of FIG. 5;

(8) FIG. 7: Alternative examples of PNA molecules tested;

(9) FIG. 8: Results of the intensity measured for the PNA molecules of FIG. 8;

(10) FIG. 9: Results of the intensity measured for alternative embodiments of the PNA molecules;

(11) FIG. 10: Examples of PNA molecules tested using a different support;

(12) FIG. 11: Results of the intensity measured for the PNA molecules of FIG. 10.

(13) FIG. 12: A scheme representing two alternative methods with which to test PNA probes: A) Chemiluminescent approach (using a microplate reader); B) Fluorometric approach (exploiting flow cytometry).

(14) FIG. 13: Performances of the PNA oligomers described in Table 1 (see below). (+) positive signal—oligo 122 (15 nM). (−) negative signal—water as control.

(15) FIG. 14: Flow cytometry analyses of the PNA oligomers described in Table 2 (see below). A) Microsphere labelling was conducted using microspheres containing the PNA oligomer DGL 21_6.0, SMART-A-Nucleobase-Biotin and Oligo DNA 21 (15 nM); B) As negative control water (no labelling).

(16) FIG. 15: Flow cytometry analyses of PNA oligomers reported in Table 2 (see below). Microsphere labelling was conducted using microspheres containing the PNA oligomer DGL 21_2.0 (A) and DGL 21_3.0 (B), SMART-A-Nucleobase-Biotin and oligo DNA 21 (15 nM).

(17) FIG. 16: PNA molecules for circulating microRNA 122. Six PNA Oligomers with sequence of nucleobases complementary to the mature miRNA122 strand. Oligomers contained unmodified PNA monomers (open circles) and monomers containing chiral modifications at gamma-positions a moiety containing a carboxylic acid group (filled circles). Either filled and empty ellipses show the abasic unit respectively with or without modifications at gamma positions.

(18) FIG. 17: Three PNA molecules for circulating microRNA 21. See FIG. 16 for the legend.

EXAMPLES

(19) Preparation

(20) Monomer Preparation

(21) Gamma-modified nucleobase-containing and “blank” PNA monomers of the present invention were prepared.

(22) In this embodiment, the PNA monomers were based on a (L) Glutamic amino derivative.

(23) In this embodiment, a nucleobase-containing gamma-modified PNA monomer (A8) and a “blank” gamma-modified PNA monomer (A9) were prepared as follows.

(24) In a first step, a gamma-modified compound of formula (A7) was prepared by 1) oxidation and 2) reductive amination of (L) Fmoc-Glutamol of formula (A6). The alcohol was oxidised to aldehyde (IBX or DMP), which was then reacted with glycine methyl ester (A2) under reductive amination conditions to yield the gamma-modified compound (A7).

(25) In a second step, the gamma-modified compound (A7) was reacted to yield either a nucleobase-containing PNA monomer (A8) or a “blank” PNA monomer (A9).

(26) To prepare a nucleobase-containing PNA monomer (A8), the gamma glutamic compound (A7) was coupled a nucleobase (A, C, G, T) and then treated under hydrolysis conditions to yield the gamma-modified nucleobase-containing PNA monomer (A8).

(27) To prepare the chiral “blank” PNA monomer (A9), the secondary amine of the compound (A7) was protected with a protective group, in this embodiment Boc, and the resulting product treated under hydrolysis conditions to obtain the “blankPNA” monomer (A9).

(28) This is illustrated in the reaction schemes of FIG. 1.

(29) PNA Molecules

(30) Various PNA molecules, including PNA oligomers containing and/or derived from monomers A8 and A9 above, were prepared and assessed.

(31) In order to assess the specificity and sensitivity of the PNA molecules, a SMART C Nucleobase modified with a biotin was used. A colorimetric approach (as shown in the scheme of FIG. 2) was performed using PNA probes supported on nylon membranes through an amino-pegylated group at their N-terminal end.

(32) In this method, DestiNA probes (from 10 to 100 μM solutions) modified at their N-terminal with an amino-pegylated group were immobilised onto Immunodyne ABC (activated nylon 6.6 membranes from Pall). DestiNA master mix was prepared with 30 μL of SMART-C-PEG-Biotin (SMART “Reader Base”) (100 μM), 45 μL of nucleic acid strands (KWT DNA and K12S DNA) at 100 nM or 45 uL PCR KWT and K12S and 20 μL of reducing agent, sodium cyanoborohydride (15 mM) and 205 μL of citrate buffer 0.1M with 0.1% SDS (pH 6). DestiNA master mix was then added onto the spotted membrane and incubated at 41° C. for 20 to 30 min. The membranes were then washed twice with 0.5× saline sodium citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) using a vacuum manifold. The membrane was blocked with a blocking solution with BSA and casein for 5 min and then, streptavidin-alkaline phosphatase solution was added onto the membrane and incubated at 29° C. for 5 min. The membranes were then washed four times with tris-HCl 0.1M/tween 20 0.5% using a vacuum manifold. Finally, NBT/BCIP chromogenic solution was added and incubated at 36° C. for 8 min. Following washing steps, photographs of the membranes were taken and the intensity of the response measured. Images were taken by a LifeCam assisted with LED illumination. Signal intensities were measured by a densitometry image software. Signal values are arbitrary units related to biotin marker intensities within the same membrane.

(33) a) PNA Molecules Containing 6 Gamma-Modified Units, and “Unmodified” Abasic Position

(34) Four different PNA probes (shown in FIG. 3) containing 6 gamma-modified units (represented in black) with an “unmodified” abasic position were immobilised on nylon membranes and tested.

(35) In particular, the repeat units shown in white were devoid of any substituent at the gamma position, and the repeat units shown in black had at their gamma position a moiety containing a carboxylic acid group.

(36) Specificity and Sensitivity of the probes were assessed as follows:

(37) (I) SPECIFICITY: the highest signal differences between two PNA probes, when reacted with: (a) KWT DNA (positive control—portion of the nucleic acid strand which contains as the nucleotide to be characterised a “G”; this nucleotide is thus characterised by means of the detectable tag carried by the modified nucleobase, in this case, following Watson and Crick pair ruling, the modified base is “C”) or (b) K12S DNA (negative control—portion of the nucleic acid strand which contains as the nucleotide to be characterised a “A”; this nucleotide should thus not be characterised by means of the detectable tag carried by the modified nucleobase as its complementary modified base “T” does not carry a detectable tag).

(38) (ii) SENSITIVITY: signals given by the four probes tested 12SRC-12DAV-13SRC-13DAV should be similar when using DNA KWT.

(39) The specific structure of the KWT DNA positive control and of the K12S negative control DNA; and the results of the signals measured for each probe, are shown in FIG. 4.

(40) In FIG. 4, DNA from PCR amplification and PNA (DGL) sequences are aligned. Mismatch nucleotides are underlined. Probes were spotted at 100 μM.

(41) It was observed that, specificity (signal different between KWT and K12S membranes) was very good for all four probes except K13SRC. It was also observed that sensitivity (difference in signal intensity provided by probes K12SRC and K13SRC if compared to K12DAV and K13DAV within the same membrane) was not satisfactory (76-90 vs 44-44) and was difficult to predict and understand based on the sequences shown in FIG. 3.

(42) b) PNA Molecules Containing Alternative Variants at the Abasic or “Blank” Position

(43) The specific structures of the probes immobilised on nylon membranes and tested as per FIG. 2, are shown in FIG. 5 below. In this case, the probes were spotted at 50 μM (half the concentration of the 6-neg probes tested in FIG. 4).

(44) In FIG. 5, Glu probes have standard glutamic acid units at both C and N terminal ends. U and Glu correspond to an abasic unit devoid of any substituent at the gamma position. CBU corresponds to an abasic unit comprising a neutral (methyl) group at its gamma position. CBC corresponds to an abasic unit having at its gamma position a moiety containing a carboxylic acid group.

(45) The results of the signals measured for each probe are shown in FIG. 6.

(46) It was observed that CBC probes (in which the unit at the abasic position contains a carboxylic group) exhibited very similar intensities between the four different probes (K12SRC, K12DAV, K13SRC and K13DAV) while keeping an acceptable level of specificity. However, the signal intensities were relatively low.

(47) It was also observed that the other probes suffered from high levels of variation in terms of intensity between the 4 types of probes.

(48) c) PNA Molecules Containing Alternative Variants at the Abasic or “Blank” Position, as Well as Variants in Other Units Containing a Nucleobase

(49) The specific structures of the probes immobilised on nylon membranes and tested as per FIG. 2, are shown in FIG. 7 below. The probes were spotted at 10, 20 and 50 μM.

(50) The results of the signals measured for each probe are shown in FIG. 8.

(51) It was observed that 3RCC configuration was the best for PNA K12SRC and K12DAV in terms of specificity (55 vs 3 and 55 vs 5, respectively). In this configuration, the blank position is separated on each side from a nucleobase unit having at its gamma position a moiety containing a carboxylic acid group by a nucleobase unit devoid of any substituent at the gamma position. This configuration also allowed signals to be of similar intensity, unlike the results obtained with 6Neg PNA structures. It was also observed that the probes performed better when the nucleobase units having at their gamma position a moiety containing a carboxylic acid group was not immediately adjacent the abasic chiral unit (3CC probes).

(52) It was also observed that the standard probes (U and Glu probes which only have negative charges through their natural amino acid glutamic groups at both C and N ends) gave lower specificity, since their triggered a higher signal when using the negative control DNA K12S.

(53) Probes K13SRC and K13DAV were then tested with the 3RCC configuration. The probes were spotted at 20 μM. The results are shown in FIG. 9.

(54) It can be seen from FIG. 9 that the 3RCC modifications offered superior performance both in respect of sensitivity and specificity.

(55) d) Use of Bead Support as an Alternative to Membrane

(56) Probes DGL-13SRC-CBC and DGL-13SRC-3RCC (see FIG. 10 below) were covalently bound to magnetic microspheres instead of nylon membranes and SMART nucleobase incorporation detected via electrochemical detection rather than colorimetry.

(57) Probes were coupled to Dynabeads Carboxylic Acid microspheres (ThermoFisher Scientific, US) using standard carbodiimide coupling chemistry in two steps. The microspheres were washed (×2, 0.02% Tween-20, 200 μL and ×2, 0.1% SDS, 200 μL), resuspended in water (˜1 million microspheres per 100 μL) and diluted in 2× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) with the pH adjusted to 6.0 (buffer A) (100 microspheres per μL). 23.5 μL of Buffer A, 12.5 μL of the functionalised microspheres (dispersed in Buffer A, containing 100 microspheres per μL), 4 μL of SMART-C-PEG-Biotin (500 μM), 7.5 μL of either s-miRNA122 or controls s-miRNA21 and s-mRNA122-A (at 1 μM, 100 nM, 10 nM or 1 nM) and 2.5 μL of reducing agent, sodium cyanoborohydride (20 mM) were added in a 200 μL eppendorf, vortexed and incubated (41° C. for 30 min, thermal cycler). The microspheres were then washed twice with Buffer A, re-suspended (in 50 μL of Buffer A), followed by addition of 10 μL of Streptavidn-HRP for 10 min. Following washing steps (3 min magnetic separation) hydroquinone (HQ) was used as electron transfer mediator and oxygen peroxide (H.sub.2O.sub.2) as HRP substrate. At this stage, amperometric measurement using a Screen-printed carbon electrodes (SPCEs) (Dropsens, Spain) was performed.

(58) The results are shown in FIG. 11.

(59) It was observed that a DGL-13SRC-3RCC probe (gamma-modified unit at blank position separated on each side from a gamma-modified nucleobase unit having a moiety containing a carboxylic acid group by a nucleobase unit devoid of any substituent at the gamma position) improved 3-fold the specific signal generated, as compared with a DGL-13SRC-3CBC probe (gamma-modified unit at blank position with no gamma-modified nucleobase units).

(60) The above experiments demonstrate the advantages of power of combining chiral modifications both at the abasic unit and different positions in the PNA probe.

(61) While the above tests show superior performance for probes having gamma-modified unit at blank position separated on each side from a gamma-modified nucleobase unit having a moiety containing a carboxylic acid group by a nucleobase unit devoid of any substituent at the gamma position, it will be appreciated that the specific position of the gamma-modified nucleobase units in the probe may be altered without compromising the improved performance of the probe. For example, the gamma-modified nucleobase units may be located two, three or four units away from the blank position. The optimal location of one or more gamma-modified nucleobase units may depend on a number of factors, such as the length of the probe, and the specific application for the probe.

(62) e) Use of Chiral Modified-PNA Probes for Circulating microRNA (miRNA) Detection with a Chemiluminescent Platform (Microplate Reader).

(63) PNA oligomers containing and/or derived from monomers A8 and A9 above, were designed/prepared to allow anti-parallel hybridisation with mature miRNA122 strands (Table 1). As shown in Table 1 (see below), PNA oligomers containing unmodified PNA monomers were synthesised, such as the probes DGL122_3.0-5.0 and some other probes carrying three or more gamma-modified units (DGL122_1.2, 4.1, 4.2).

(64) In order to test the PNA molecules, modified nucleobases tagged with Biotin (SMART-C-Nucleobase-Biotin) were used along with a chemiluminescent method (as shown in the scheme of FIG. 12A).

(65) PNA molecules were tested to assess the specificity and sensitivity of the PNA molecules in relation to: i) the presence of gamma-modified units; ii) the presence of gamma-modifications within the abasic unit; iii) abasic unit position and length of the PNA oligomers (see Table 1 below).

(66) In this method, probes modified at their N-terminal with an amino-pegylated group were covalently bound to carboxylated Dynabeads® (M-270 Carboxylic Acid) using a two-step protocol (without NHS) carbodiimide coupling chemistry according to the manufacturer protocol (Thermo Fisher Scientific).

(67) A master mix was prepared with 50 μL of microspheres (4×10.sup.4 beads/uL), SMART-C-Nucleobase-Biotin (Reader Base) (2 μM), nucleic acid strands (Oligo DNA 122) at 15 nM or water instead as control and reducing agent, sodium cyanoborohydride (1 mM) and SCD buffer (2×SSC and 0.1% SDS-pH 6.0) up to a final volume of 50 μL. DestiNA master mix was then vortexed and incubated at 41° C. for 1 h in a thermal cycler. Upon completion of the reaction, the microspheres were washed three times with 200 uL of washing Buffer A (PBS-Tween 0.1%). The microspheres were then pelleted and the supernatant removed and incubated for 5 min at RT with 100 uL of Pierce High Sensitivity Streptavidin-HRP (1:8000) solution (Thermo Fisher Scientific). Following washing steps (4× in Buffer A), the microspheres were pelleted and re-supernatanted in 100 uL of substrate—SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The microspheres were incubated for 5 min at RT. Upon completion of the incubation, the microspheres/substrate was transferred (100 uL) to the substrate (a white 96-well plate) for final reading using a plate reader with chemiluminescence detection capability (FLUOstar Omega) (FIG. 12A). The results are shown in FIG. 13.

(68) The best performance results were obtained with capture probes 122_1.2, 122_4.1 and 1224.2. Specifically, it was observed that 122_1.2, 1224.1 and 122_4.2 probes with either a gamma-modified unit at the blank position (a gamma modified abasic position) or distributed across the PNA backbone have an improved specific signal generated, as compared with a DGL122_3.0-5.0 probes (without a gamma-modified unit at the blank position or a gamma-modified nucleobase unit across the PNA backbone).

(69) PNA oligomers 122_4.1 (not having a gamma-modified unit at the blank position and two gamma-modified nucleobases on the left side and a single gamma-modified nucleobase on the right side (see Table 1)) exhibited good performance. This shows that the performance of a PNA Oligomer (i.e. an improvement in the signal-to-background ratio; where the background is compared to that obtained when the reaction is carried out using water as control (DestiNA probes followed by “-” in FIG. 13)) is in part dependent on the gamma-modified nucleobase units' position within the probe These results confirm that the optimal location and number of gamma-modified nucleobase units may depend on a number of factors and on the specific application of the probe.

(70) f) Use of Chiral Modified-PNA Probes for Circulating microRNA (miRNA) Detection with Flow Cytometry

(71) Flow cytometry was used to further study the effect of gamma-modified units at “blank positions” as well as those distributed across the PNA oligomer's backbone (FIG. 12B).

(72) Three PNA oligomers containing and/or derived from monomers A8 and A9 above, were designed/prepared to allow anti-parallel hybridisation with mature miRNA21 strands (Table 2). PNA oligomers DGL21_2.0 and DGL21_6.0 were designed to hybridize to the same region of mature miRNA21. Both Oligomers carry two gamma-modified units respectively on the two thymine nucleobases beside (or adjacent) the blank position. Additionally, DGL21_6.0 contains a gamma-modified unit at the blank position and the sequence is slightly longer (19-mer instead of 17). The other 17-mer PNA oligomer (DGL21_3.0) was designed to allow anti-parallel hybridisation to a different region of the mature miRNA21 strand and carries two gamma-modified units on the thymine nucleobases either side of the blank position (Table 2). For the three PNA oligomers, the blank position was positioned so that post-hybridisation, the mature miRNA21 strand presents a uracil (Table 2, nucleobases shown in green) in front of the blank position thereby allowing incorporation of an adenine modified nucleobase tagged with Biotin (SMART-A-Nucleobase-Biotin).

(73) PNA oligomers DGL21_2.0, DGL21_6.0 and DGL21_3.0 were covalently bound to Dynabeads Carboxylic Acid microspheres (ThermoFisher Scientific) using standard carbodiimide coupling chemistry in two steps (coupling was performed as reported above). To assess the specificity and sensitivity of the PNA molecules, performance of microspheres was assessed by selective incorporation of SMART-A-Nucleobase-Biotin (Reader Base).

(74) The microspheres were processed using a flow cytometric method. A master mix was prepared with 50 μL of microspheres (8×10.sup.7 beads/uL), SMART-A-Nucleobase-Biotin (30 μM), nucleic acid strands (Oligo DNA 21) at 15 nM or water instead as control and reducing agent, sodium cyanoborohydride (150 μM) and Phosphate Buffer pH 6 (150 mM) up to a final volume of 50 μL. The master mix was then vortexed and incubated at 41° C. for 1 h in a thermal cycler. Upon the completion of the reaction, the microspheres were washed three times with 200 uL of washing Buffer A (PBS-Tween 0.1%). The microspheres were then pelleted and the supernatant removed and incubated for 1 h RT with 100 uL of Streptavidin-R-Phycoerythrin Conjugate—SAPE (20 μg/mL), purchased from Thermo Fisher Scientific. After SAPE incubation, microspheres were analysed by BD FACSCanto (PE-Channel, filter 585/42) and dot-plots obtained through Flowjo.

(75) The results are shown in FIG. 14.

(76) Population shifts on the PE channel were clearly seen with microspheres containing the PNA oligomer DGL 21_6.0. It was observed that the DGL 21_6.0 probe (containing the gamma-modified unit at the blank position and others distributed across the PNA backbone) enabled the efficient dynamic incorporation of the Reader Base with a specific signal generated (population shift on the PE channel) (FIG. 14), as compared with a DGL21_2.0 and DGL21_3.0_probes (without gamma-modified unit at the blank position) (FIG. 15). These results show how the adding of gamma-modification at the blank position (DGL 21_6.0) brings a superior SMART-A-Nucleobase-Biotin incorporation when compared to a pretty similar sequence but without gamma-modification at the blank position such as DGL21_2.0 (see Table 2).