WO2015071552A1 - Sondes à plusieurs motifs présentant une spécificité élevée et procédé de conception de ces dernières - Google Patents

Sondes à plusieurs motifs présentant une spécificité élevée et procédé de conception de ces dernières Download PDF

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WO2015071552A1
WO2015071552A1 PCT/FI2014/050876 FI2014050876W WO2015071552A1 WO 2015071552 A1 WO2015071552 A1 WO 2015071552A1 FI 2014050876 W FI2014050876 W FI 2014050876W WO 2015071552 A1 WO2015071552 A1 WO 2015071552A1
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unit
binding
nucleic acid
probe
target nucleic
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Petri Saviranta
Marika HEIKKINEN
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Teknologian Tutkimuskeskus Vtt
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation reaction
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/161—Modifications characterised by incorporating target specific and non-target specific sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/197—Modifications characterised by incorporating a spacer/coupling moiety
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/162—Helper probe

Definitions

  • the invention relates to multi-unit nucleotide probes capable of discriminating highly homologous nucleic acid sequences from each other and to a method of designing such probes.
  • Nucleic acid probes recognizing two distinct non-continuous regions of a target nucleic acid have been suggested for various purposes.
  • international patent publication WO 96/17955 employs hybritope mapping for identifying high efficacy binding sites in a target DNA sequence to design a strongly binding discontinuous probe with two or more binding sequences connected directly or with an organic linker molecule.
  • a pool of discontinuous probes is constructed and screened for identifying the discontinuous probe with the highest degree of binding or the fastest hybridization rate as the best candidate probe.
  • the purpose is to combine the strongest or fastest-binding sequences into a single discontinuous probe.
  • Gentalen Nucleic Acids Res. 1999, 27:1485-1491
  • Bates Anaal Biochem. 2005, 342:59-68
  • one microarray spot contains a mixture of two oligonucleotides which bind the target nucleic acid at different sites.
  • This approach overcomes the oligonucleotide length limitation of photolithographically manufactured microarrays (e.g. Affymetrix) by utilizing the cooperative binding of two relatively short oligonucleotides (25-30mer) to achieve the benefits of longer oligonucleotides (60-70mer).
  • a remaining downside is that the ratio of the component probes is prone to variances in the solid phase immobilization densities, which may be difficult to reproduce in manufacturing.
  • US 2007/0259344 discloses a multi-unit probe with at least two binding units.
  • the purpose of using multi-unit probes is to increase the number of individual probe sequence units in an array or to decrease the number of arrays or spots required for an assay. There is no attempt to exploit the co-operative binding of several probe units in a multi-unit probe.
  • WO 201 1/027966 discloses a target discriminative (TD) probe comprising a 5'-second hybridization portion that mainly determines target specificity, a separation portion, and a 3'-first hybridization portion that initiates stable hybridization.
  • the TD probe hybridizes with a target nucleic acid through both of the 5'-second hybridization portion and the 3'-first hybridization portion.
  • both the 5'-second hybridization portion and the separation portion are not hybridized with the non-target nucleic acid sequences owing to their low Tm value.
  • Nucleic acid hybridization assays commonly suffer from poor probe specificity. This is especially true in nucleic acid based pathogen diagnostics where closely related viruses or bacteria need to be identified; in gene expression studies where the presence of homologous genes interferes with the analysis; and in various genotyping applications (such as HLA genotyping for tissue transplantation), where there is a multitude of homologous alleles differing only by a few single nucleotide polymorphisms (SNP). Often, it is difficult to design specific, discriminating probes for highly homologous targets, when there are only a few differences between them, and these are far apart in sequence. Even if it would be possible to make an extended probe to cover the differing sites, this would usually not result in increased specificity, because long probes tend to tolerate a few separated mismatches very well.
  • SNP single nucleotide polymorphisms
  • probes contain two or more distinct binding units, they are not specific enough for applications, such as diagnostic methods based on SNP analyses, requiring discrimination of highly homologous non-continuous nucleic acid sequences. Or, as in the case of mixed probes, their molar ratios are challenging to reproduce in manufacturing. Thus, there is an identified need in the field for more specific probes which can be reproducibly manufactured.
  • the present invention provides a multi-unit probe which binds to a target nucleic acid and comprises at least two binding units joined by at least one linking unit, wherein the multi-unit probe hybridizes stably to the target nucleic acid in stringent conditions only when all binding units bind to their respective complementary regions; and the multi-unit probe does not substantially self-hybridize or form secondary structures.
  • each binding unit of the multi-unit probe is complementary to a distinct region in the target nucleic acid and has a Gibbs free energy of binding ( ⁇ ) greater than or equal to -16 kcal/mol, as calculated in the presence of 0.9 M [Na+].
  • the combined ⁇ of all binding units is lower than -16 kcal/mol, preferably lower than -20 kcal/mol, more preferably lower than -25 kcal/mol, as calculated in the presence of 0.9 M [Na+].
  • the combined ⁇ of all binding units to a non-target nucleic acid is at least 1 .5 kcal/mol higher, preferably at least 3 kcal/mol higher, than the corresponding combined ⁇ to a target nucleic acid.
  • the multi-unit probe may hybridize to a specific human leucocyte antigen (HLA) allele.
  • HLA human leucocyte antigen
  • the present invention provides a microarray comprising a plurality of multi-unit probes of the first aspect of the present invention, wherein each multi-unit probe hybridizes to a different target nucleic acid.
  • the present invention provides a method of designing a multi-unit probe which binds to a target nucleic acid and comprises at least two binding units joined by at least one linking unit, wherein the method comprises:
  • the designing method may comprise additional features independently as follows:
  • each of the binding units has ⁇ greater than or equal to -16 kcal/mol, as calculated in the presence of 0.9 M [Na+];
  • the combined ⁇ of all binding units is lower than -16 kcal/mol, preferably lower than -20 kcal/mol, more preferably lower than -25 kcal/mol, as calculated in the presence of 0.9 M [Na+];
  • Figure 1 illustrates the basic idea of the invention and shows how a stable hybrid is only formed when the target nucleic acid contains complementary sequences for both (all) binding units of the MultiGrip probe.
  • Target A contains two complementary sequences and thus can form a stable hybrid with the MultiGrip probe.
  • Targets B and C contain one complementary sequence and Target D none, therefore they cannot form stable hybrids with the MultiGrip probe.
  • Figure 2 shows partial sequences (nucleotides 95-135 of a PCR amplicon) for a group of HLA-DQB1 alleles (upper panel) and the binding unit sequences of multi-unit probes designed for identification of the DQB1 *030x alleles (lower panel).
  • the discriminating polymorphic nucleotides within the 030x alleles are marked with arrows.
  • the dashes represent the omitted nucleotides between the binding units.
  • the omitted nucleotides are replaced by five thymidine oligomer, dT(5), linkers in the multi-unit probes.
  • Figure 3 shows partial sequences (nucleotides 34-78 of a PCR amplicon) for a group of HLA-DQB1 alleles (upper panel) and the binding unit sequences of the multi-unit probe designed for identification of the DQB1 *0601 allele (lower panel).
  • the discriminating polymorphic nucleotides are marked with arrows.
  • the dashes represent the omitted nucleotides between the binding units.
  • the omitted nucleotides are replaced by oligo(dT(5)) linkers in the multi-unit probes.
  • Figure 4 demonstrates hybridization of the DQB1 *030x allele samples to the different probe type alternatives. Binding units A and B are spotted either as separate short probes (A and B), mixed (A+B mix), as multi- unit probes (AB MultiGrip) or as part of long probes (AB long). The hybridization results are shown as percentage values relative to the allele- specific multi-unit probe signal, after normalization towards the control probe. Single unit probes are not strong enough to hybridise in these reaction conditions. Mixed and multi-unit probes, however, bind to their specific target samples. With the multi-unit probes the signal levels are 3-7 times higher than with mixed probes, producing higher signal-to-background ratios and therefore higher sensitivity and increased specificity. The long probes bind to all of the allele samples with high signal intensities, showing little if any discrimination between the alleles.
  • Figure 5 demonstrates hybridization specificity of the different probe type alternatives to the DQB1 * 030x allele samples.
  • the data from Figure 4 is herein presented for each probe type as percentage value relative to the allele- specific signal. Cross-reactivity between the samples is lower with multi-unit probes than with any other probe type, remaining below 25 % in all cases.
  • Figure 6 demonstrates hybridization of the selected HLA-DQB1 allele samples to the DQB1 * 030x multi-unit probes. All of the multi-unit probes allow the correct identification of their corresponding target alleles. Cross- reactivity between the samples remains below 25 % in all cases. The hybridization results are shown as percentage values relative to the allele- specific multi-unit probe signal, after normalization towards the control probe.
  • Figure 7 shows partial sequences (nucleotides 22-1 19 of a PCR amplicon) for a group of HLA-DRB1 * 04xx alleles (upper panel) and the binding unit sequences of the multi-unit probe designed for identification of the DRB1 * 0403 and * 0406 alleles (lower panel).
  • the discriminating polymorphic nucleotides are marked with arrows.
  • the dots represent the omitted nucleotides between the binding units.
  • the omitted nucleotides are replaced by oligo(dT(5)) linkers in the multi-unit probes.
  • Figure 8 demonstrates hybridization of a group of HLA-DRB1 * 04xx allele samples to the DRB1 * 0403/6 multi-unit probe. Some of the samples had purified DNA as a starting material for PCR amplification and others had dried blood spots. In all cases the multi-unit probe allows correct identification of the corresponding target alleles without cross-reactivity from unspecific samples. The hybridization results are shown as normalized signals towards the control probe.
  • the present invention provides a multi-unit probe which binds to a target nucleic acid through at least two binding units complementary to discontinuous, predetermined target motifs in the target nucleic acid.
  • the binding units are designed such that stable hybridization occurs in stringent conditions only when all binding units bind to their respective complementary regions, i.e. the target motifs, in the target nucleic acid.
  • the basic idea of the invention is illustrated in Figure 1 .
  • multi-unit probe and “MultiGrip probe” are interchangeable.
  • TwinGrip probe refers to a multi-unit probe with two binding units
  • TripleGrip probe refers to a multi-unit probe with three binding units.
  • target nucleic acid refers to any nucleic acid molecule the presence of which in a biological sample is to be determined.
  • the biological sample may be, for instance, a blood sample, such as a whole blood sample, or bodily fluid such as amniotic fluid or cerebrospinal fluid, or any tissue sample, such as a tissue biopsy, a lymph node biopsy, or a biopsy from a tumor lesion.
  • tissue sample such as a tissue biopsy, a lymph node biopsy, or a biopsy from a tumor lesion.
  • Other non-limiting examples of the biological sample include sputum, mucus, urine, stool or any other body excretion.
  • the biological sample may be pretreated in a suitable manner, including but not limited to nucleic acid extraction, known to those skilled in the art, prior to the determination of the presence or absence of the target nucleic acid in said biological sample.
  • the target nucleic acid may be any nucleic acid sequence, such as a gene sequence, a regulatory sequence near a gene, or any other coding or noncoding region of the genome, possibly with a disease association.
  • a non- limiting example of a genome region with a disease association is the HLA (human leucocyte antigen) gene region, with allelic variants linked to risk of Type 1 Diabetes.
  • the target nucleic acid may be pathogenic DNA or RNA.
  • target motif refers to a pre-selected region in the target nucleic acid, against which the present probe ' s respective complementary binding unit has been designed.
  • target motifs are separated in nucleotide sequence by at least about 10 nucleotides.
  • At least two target motifs are to be selected for each desired target nucleic acid to allow designing and producing a multi-unit probe according to the present invention. At least one of the target motifs or a combination of the target motifs must be unique for the desired target nucleic acid. This allows the present probes to discriminate highly similar target and non-target nucleic acids from each other.
  • the difference(s) between a target and a non-target nucleic acid are based on polymorphism(s) such as single nucleotide polymorphism(s) (SNPs).
  • the target motif(s) may be selected such that the probe is able to discriminate different gene alleles from each other.
  • discriminative region refers to a nucleotide sequence region in a target molecule or a corresponding nucleotide sequence region in a highly similar non-target molecule wherein the corresponding nucleotide sequence regions in the target molecule and the non-target molecule differ from each other by at least one nucleotide.
  • the terms "highly similar target and non-target nucleic acids” and “closely related target and non-target nucleic acids” may be used interchangeably. These terms refer to target and non-target nucleic acids which have similar but not identical nucleotide sequences.
  • the percentage of identical nucleotides in the sequences of highly similar target and non-target nucleic acids can be greater than 80%, greater than 90%, greater than 95% or even greater than 99%.
  • the first step of a method of designing a multi-unit probe according to the present invention is to identify at least one discriminating region in a desired target nucleic acid. This may be achieved by downloading nucleic acid sequences of a target and a related non-target nucleic acid from publicly available databases, such as NCBI's (National Center for Biotechnology Information) GenBank. Readily available computational programs may be used to align the sequences in order to find conserved homologous areas and, more importantly, variable sites therein. Notably, more than one target nucleic acid sequence and/or non-target nucleic acid sequence may be downloaded and aligned simultaneously.
  • NCBI's National Center for Biotechnology Information
  • the identified variable sites are then screened for unique nucleotide differences, which discriminate the target nucleic acid(s) from the non-target nucleic acid(s). This may be performed by screening through the sequences systematically, nucleotide by nucleotide.
  • the discriminating region may involve any nucleotide change including one or more substitutions, deletions, insertions, and/or inversions as understood by a skilled person.
  • the discriminating region is a polymorphic site either with a single nucleotide polymorphism (SNP) or with several closely located polymorphic nucleotides within a total span of less than about 20 nucleotides.
  • the target nucleic acid(s) cannot be uniquely identified by any single polymorphic site, a combination of two or more polymorphic sites can be chosen which is unique to the allele in question.
  • the distance between the individual polymorphic sites can be 10 to 100 nucleotides, preferably 20 to 40 nucleotides.
  • At least one of the binding units of a multi-unit probe according to the present invention is designed to be complementary to a discriminating region identified as described above.
  • the term "complementary” is well known in the art and it means Watson-Crick base pairing where nucleobase adenine (A) in a target motif sequence is represented by nucleobase thymine (T) in a corresponding binding unit, or vice versa. Accordingly, nucleobase cytosine (C) in a target motif is represented by nucleobase guanine (G) in a corresponding binding unit, or vice versa.
  • the complementary sequence to, for instance, 5"-T-T-C-A-G-3" is 3'-A-A-G-T-C-5".
  • each individual binding unit is designed such that its binding strength as a single unit is insufficient for stable binding with the target nucleic acid in a given stringent hybridization conditions. Only co-operative binding of all binding units enables stable binding of the probe to its target nucleic acid. Even a single mismatch between a binding unit and a respective target motif in a non-target nucleic acid lowers the overall binding strength of the probe enough to prevent stable binding in the given stringent hybridization conditions. Even alleles or other nucleic acid molecules differing solely by an SNP are distinguishable by the present multi-unit probes. Further, the present probes are specific and sensitive enough to allow detection of not only homozygotic genotypes but also heterozygotic ones.
  • hybridize refers to the physical interaction between complementary regions of two single-stranded nucleic acid molecules creating a double-stranded structure, also termed as a heteroduplex.
  • hybridize refers to interactions between target motifs and corresponding binding units under hybridization conditions that allow complementary regions of the two molecules to interact by hydrogen bonding and remain engaged.
  • hybridization conditions refers independently not only to the conditions of the hybridization step per se, but also to the conditions of one or more washing steps performed thereafter.
  • Modifiable variables of the hybridization conditions include, but are not limited to, duration (typically from a few minutes to a few days), temperature (generally from 37°C to 70°C), salt composition and concentration (e.g., 2- 6xSSC, or SSPE), chaotropic agent composition (e.g., formamide, or dimethyl sulfoxide (DMSO)) and concentration, and usage of substances that decrease non-specific binding (e.g., bovine serum albumin (BSA), or salmon sperm DNA (ssDNA)).
  • BSA bovine serum albumin
  • ssDNA salmon sperm DNA
  • hybridization stringency refers to the degree to which mismatches are tolerated in a hybridization assay. The more stringent the conditions, the more likely mismatched heteroduplexes are to be forced apart, whereas less stringent hybridization conditions enhance the stability of mismatched heteroduplexes. In other words, increasing the stringency increases the specificity of the hybridization reaction. A person skilled in the art is able to select the hybridization conditions such that a desired level of stringency is achieved. Generally, the stringency may be increased by increasing temperatures (closer to the melting temperature (Tm) of the heteroduplex), lowering the salt concentrations, and using organic solvents. As known in the art, stringent hybridization conditions are sequence dependent and, thus, they are different under different experimental parameters.
  • hybridization conditions can be chosen such that a single mismatch renders a heteroduplex unstable. Such hybridization conditions may be called as "highly stringent hybridization conditions".
  • the Tm is the temperature (under defined ionic strength, pH, and DNA concentration) at which 50% of the target motifs are hybiridized with their matched binding units. Stringent conditions may be obtained by performing the hybridization in a temperature equal or close to the Tm for the probe in question.
  • Exemplary stringent hybridization conditions for short binding units include 6xSSC, 0.5% Tween20, and 20% formamide incubated at 37°C in 600 rpm for one hour, followed by washing twice in TBS buffer containing 0.05% Tween20 at room temperature.
  • the second step of a method of designing a multi-unit probe according to the present invention is to design at least two binding units, at least one of which is to be designed around a discriminating site identified in step one, such that the multi-unit probe hybridizes stably to the target nucleic acid in stringent conditions only when all binding units bind to their respective complementary regions, i.e. the target motifs.
  • an optimal binding strength for each of the binding units enabling stable hybridization of the multi-unit probe in stringent conditions only when all binding units bind to their respective target motifs, is to be determined by experimental trials with different sequence and length variations at different hybridization washing conditions.
  • the binding strength of individual binding units is to be estimated by thermodynamic calculations.
  • a suitable parameter for such purposes is the Gibbs free energy of binding, denoted as AG. The lower, i.e. more negative, the AG value, the stronger the binding.
  • thermodynamic calculations are based on the probe's and target motifs nucleic acid sequences and published, experimentally determined nucleotide pairing parameters (see e.g. SantaLucia J Jr (1998): A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 95: 1460-5).
  • the calculations may be performed automatically using publicly available computer programs or web based tools, such as the DINAMelt Web Server (Markham, N. R. & Zuker, M. (2005) DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res., 33, W577-W581 ).
  • DINAMelt Web Server Markham, N. R. & Zuker, M. (2005) DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res., 33, W577-W581 .
  • the free energy of binding AG
  • the AG calculations are salt concentration dependent. Herein, all calculations have been performed using an assumed [Na+] of 0.9 M, unless otherwise stated. If different salt concentrations are used, the AG-based guidelines described herein need to be adjusted accordingly.
  • candidate binding units For each selected discriminating site on the target nucleic acid, several candidate binding units are to be initially designed by varying the number of nucleotides on both sides of the discriminating nucleotide(s). The candidates may then differ both in length and in nucleotide composition (e.g. %GC), depending on the nucleotide sequence in the target motif site. It is also possible to use modified nucleotides (e.g. LNA) in the binding unit, if their thermodynamic parameters are available for the calculation of the binding strength.
  • modified nucleotides e.g. LNA
  • the AG values are to be calculated both for the perfectly matched duplex (formed with the desired target motif) as well as for any relevant, partially matching, potentially cross-reactive nucleic acids (the non-desired target motifs).
  • binding units suitable for being comprised in the present multi-unit probes should abide by the following rules:
  • the AG of binding to a desired individual target motif should be > -16 kcal/mol in order to prevent the multi-unit probe from binding through any solitary binding unit;
  • the AG of binding to a non-desired target motif should be at least 1 .5 kcal/mol higher than the AG for the desired target motif.
  • the length of the binding units fulfilling the above AG criteria is around 10 to 20 bases.
  • the combined binding strength (AGcombined) of a multi-unit probe may be roughly estimated as the sum of the AG values of the individual binding units. This is just a rough approximation, as it does not take into account the effect of one or more linking units present in the multi-unit probe, or the distance between the target motifs on the target strand. Nevertheless, the AGcombined value may be used for the comparison of different multi-unit probe candidates, before actual synthesis and testing of the best candidates.
  • multi-unit probes according to the present invention comply with at least one of the following rules:
  • the AGcombined to the desired target nucleic acid molecule should be at least ⁇ -16 kcal/mol, more preferably ⁇ -18 kcal/mol, the most preferably ⁇ -20 kcal/mol;
  • the AGcombined to any non-target nucleic acid molecule should be preferably > -20 kcal/mol, more preferably > -18 kcal/mol, the most preferably > -16 kcal/mol;
  • the AGcombined for the non-target nucleic acid should be at least 1 .5 kcal/mol higher, more preferably at least 3 kcal/mol higher, than the
  • oligonucleotide probe design principles apply for the individual binding units of the multi-unit probes, as well as for the multi-unit probe as a whole. These principles have been described in multiple research papers, and a person skilled in the art is readily able to apply them for the present multi-unit probes. These principles include, but are not limited to, the avoidance of internal secondary structures, either within a single binding unit, or through complementarity between different binding units in the same multi- unit probe. Moreover, also the self-complementarity between two probe molecules should be avoided, as this may lead to probe-probe hybrids which are unavailable for binding to the target.
  • the present multi-unit binding probes do not substantially self-hybridize or form secondary structures
  • the third step of a method of designing a multi-unit probe according to the present invention comprises assessing the level of possible self-hybridization and formation of secondary structures, and accepting only probes which do not substantially self-hybridize or form said secondary structures.
  • unwanted secondary structures have a ⁇ not less than -5 kcal/mol, preferably not less than -2 kcal/mol.
  • the final evaluation of the performance of the present multi-unit probe may be done by testing in an actual assay set-up.
  • cross-reactivity between the present multi-unit probe and an unspecific nucleic acid molecule should be less than 25 %, preferably less than 10 %.
  • cross-reactivity means that besides hybridizing with the probe ' s specific target nucleic acid molecule, the probe can bind to also other, unspecific targets. Regardless of highly stringent conditions in hybridization reaction this unspecific binding to undesired target molecules is strong enough to form a detectable stable hybrid.
  • the percentage value of cross-reactivity can be calculated as ratio between normalized signals from specific and unspecific target binding.
  • the chemical composition of the binding units in the present multi- unit probes may be e.g. DNA, RNA, locked nucleic acid (LNA), or peptide nucleic acid (PNA), or they may be composed of mixed polymers containing any number of monomers of DNA, RNA, LNA, PNA, or other nucleic acid analogues.
  • the "linking unit” means a chemical entity which connects two or more binding units together. In a typical multi-unit probe configuration, the binding units and the linking units alternate in a linear order such that for a multi-unit probe with n binding units there are n-1 linking units.
  • the linking unit may, for example, covalently connect the 3'-end of one binding unit to the 5'-end of the next binding unit.
  • a linking unit may also connect more than two different binding units together in a multivalent configuration.
  • Suitable linking units include, but are not limited to, non-targeting oligonucleotide chains (e.g. oligo(dT)), poly(ethylene glycol) (PEG), and aliphatic carbon chains.
  • the linking unit may act as a spacer between the binding units, reducing steric hindrance and conformational constraints when the binding-units bind to their respective discontinuous target motifs.
  • the length of an oligo(dT) linking unit may vary from 0 to 30 bases, a preferred length being from 4 to 10 bases.
  • two binding units may be directly joined together through a phosphodiester bond between the 3' end of the one binding unit and the 5'-end of the other binding unit, without any intervening linking unit.
  • Hybridization of the present multi-unit probe to its target nucleic acid may be detected using any means known in the art. Accordingly, either the probe or the target nucleic acid may be coupled directly or indirectly with any available label-containing moiety, such as a fluorescent label (e.g., Cy5, Cy3, Cy2, TexasRed, FITC, Alexa series of dyes, TMR, FluorX, ROX, TET, or HEX), a radioactive label (e.g., 32P, 33P, or 33S), or an enzyme (e.g.
  • a fluorescent label e.g., Cy5, Cy3, Cy2, TexasRed, FITC, Alexa series of dyes, TMR, FluorX, ROX, TET, or HEX
  • a radioactive label e.g., 32P, 33P, or 33S
  • an enzyme e.g.
  • Luciferace horseradish peroxidase, alkaline phosphatase, or beta-galactosidase used for catalyzing a chemiluminescent reaction with substrates like Tetramethyl Benzidine, 4-Nitrophenyl phosphate, or Fluorescein di- -D-galactopyranoside).
  • substrates like Tetramethyl Benzidine, 4-Nitrophenyl phosphate, or Fluorescein di- -D-galactopyranoside.
  • a person skilled in the art is able to select a suitable detection means and method for each selected label-containing moiety. For instance, colorimetric detection may be used for biotin-streptavidin-enzyme conjugates. Hybridization may also be detected using applications in which no label is needed, such as those in which the detection is based on electric impulses (e.g., Motorola eSensorTM).
  • the present multi-unit probes may be provided attached onto the surface of a solid support having any desired size, varying typically from a couple of millimeters to a few centimeters.
  • a preferred type of a solid support is a microarray format generally known in the art.
  • the solid support may be made, for instance, of glass, plastic, metal, silicon, silicon oxide, silica, or a combination thereof.
  • the solid support may require a surface treatment, such as coating with aminosilane or epoxysilane.
  • the probes may be attached to the solid surface by any method known in the art including, but not limited to, covalent and non-covalent binding.
  • the probes may be attached to the surface as amino modified oligonucleotides, e.g. through a C6 linker with an amino group coupled to the probe's 5' end.
  • the surface attaching process may be performed manually by pipetting or robotically by printing, or spotting, with any commercially available arrayer suitable for this purpose (e.g. Qarray2 by Genetix).
  • the probes may be synthesized in situ directly onto the solid support by any available method, such as ink-jet technology or photolithography.
  • the microarray may be analyzed by any equipment applicable to this purpose and suitable for detecting the label type in question.
  • a non-limiting example of a device designed for the detection of fluorescent signals is Tecan LS400 Confocal Laser Scanner.
  • the present multi-unit probes may be used for a variety of different purposes including, but not limited to, diagnostic purposes.
  • the present probes may be designed to discriminate between allelic variants of a gene e.g. in cases where different allelic variants predispose to or cause a disease or a pathogenic condition at different risk levels.
  • haplotypes i.e. the presence of two or more single nucleotide polymorphisms (SNPs) in cis (in the same chromosome) instead of in trans (in different chromosomes).
  • SNPs single nucleotide polymorphisms
  • the multi-unit probe can be used to recognize the cis orientation, as it will only hybridize to the target when both (all) SNPs reside in the same target molecule.
  • the present probes may be designed for and utilized in pathogen diagnostics, such as virus and bacteria diagnostics.
  • pathogen diagnostics such as virus and bacteria diagnostics.
  • a multi-unit probe may be designed to hybridize to a target nucleic acid only when the right pattern of two or more polymorphic sites is present. This will be more economical than designing several probes to look at each polymorphic site individually.
  • the multi-unit probe will only hybridize to targets which contain all the polymorphic sites in the same molecule (and not in two or more different molecules, which could be the case in a mixed infection).
  • the multi-unit probes can also be used to increase the specificity of hybridization between two closely related sequences.
  • the increased specificity can be achieved by designing the individual binding units only to the discriminative regions, even if they are far apart in the sequence.
  • the binding strengths of the individual units are designed such that all binding units are required for a stable hybridization between the multi-unit probe and the target.
  • the co-operative use of more than one discriminatory region simultaneously will then result in a compounded effect in the total discriminatory power of the multi-unit probe.
  • a three-unit probe could be designed to simultaneously look at three SNPs which are located at nucleotide positions 50, 150 and 250 in the target nucleotide sequence.
  • the hypothetical three-unit probe could consist of three 12-nucleotide binding units joined together by two 5-nucleotide linking units, resulting in a total length of 46 nucleotides and 36 target-hybridizing nucleotides.
  • a conventional probe sequence spanning the same three SNPs would need to be at least 201 nucleotides long.
  • each of the 12- nucleotide binding units would encounter one mismatched nucleotide, whereas the long conventional probe would be able to hybridize with the target using two 99-nucleotide stretches of perfectly matching sequence.
  • this hypothetical example illustrates the benefits of using multi-unit probes to increase the specificity of hybridization in situations where the target and non-target nucleic acids contain only a few polymorphic nucleotides relatively far apart from each other.
  • "far apart” may mean any distance more than approximately 10 nucleotides, up to hundreds or thousands or tens of thousands of nucleotides or more, depending on the particular system and hybridization conditions.
  • Non-limiting examples of multi-unit probes according to the present invention include probes detecting allelic variation in the HLA gene region, particularly in the HLA-DQB1 , HLA-DQA1 , and HLA-DRB1 loci associated with susceptibility to Type 1 Diabetes. Thermodynamic values of herein-described HLA probes are shown in Tables 1 and 2 below.
  • multi- unit probes have been designed against the HLA-DQB1 * 03 allele group.
  • a probe for 0301 allele comprises a first and a second binding unit comprising nucleic acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:3, respectively.
  • a probe for 0302 allele comprises a first and a second binding unit comprising nucleic acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively.
  • a probe for 0303 allele comprises a first and a second binding unit comprising nucleic acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:3, respectively.
  • a probe for 0304 allele comprises a first and a second binding unit comprising nucleic acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:4, respectively.
  • a multi-unit probe have been designed for the HLA-DQB1 * 0601 allele.
  • a probe for the 0601 allele comprises a first and a second binding unit comprising nucleic acid sequences set forth in SEQ ID NO:5 and 6, respectively.
  • a multi-unit probe have been designed for the dual identification of HLA-DRB1 * 0403 and * 0406 alleles.
  • a probe for the 0403 and 0406 alleles comprises a first, a second and a third binding unit comprising nucleic acid sequences set forth in SEQ ID NO:7, 8 and 9, respectively.
  • HLA human leucocyte antigen
  • the target sequence of the present multi-unit probes was based on the same above-described SNPs but they were combined into single probes in a unique way.
  • the probes were designed to have three units, binding units A and B for discriminating between the alleles and a poly-thymidine linker connecting the A and B parts ( Figure 2).
  • Pentamer thymidine nucleotides, also referred as oligo(dT(5)) were used as a neutral linker that has virtually no binding to the target strand.
  • the precise DNA sequence and length of the probes were adjusted based on calculated thermodynamics of the probe-target strand duplex structures. This was evaluated with tools from The DINAMelt Web Server (The RNA Institute, University at Albany, State University of New York). Using the Two-state melting application the hybridization efficiencies between the designed probe and the target stand (2 nM strand concentration) were evaluated. The binding efficiency free energy ( ⁇ ) and melting temperatures (Tm) were calculated. Using the Quikfold application the energies and structures for the strand folding were calculated and evaluated. The aim was to have sufficiently high binding of the fully complementary target strand to its corresponding probe in the highly stringent conditions used in the hybridisation. Either of the single sequence units should not be binding in these conditions alone. No self-dimers or secondary structures of the probes should exist that could disturb the target binding. Multi-unit probe array printing
  • the designed multi-unit probes can be covalently attached to flat 96-well plate bottoms by any array printing method and conjugation chemistry known in the art.
  • a preferred protocol was used to covalently attach amino modified oligonucleotides (ordered from biomers.net) onto chemically modified 96-well plate bottoms using QArray2 (Genetix) array printer.
  • QArray2 Genetix array printer.
  • Each oligonucleotide was spotted robotically in an ordered array as three replicate spots per well. After spotting the wells were block with an appropriate blocker, washed twice with PBS buffer and then dried.
  • binding units A and B were also arrayed as single nucleic acid probe molecule spots to evaluate their respective binding capability to the target DNA strands.
  • long probes which contained both A and B binding units and also the nucleic acid sequence in between them, were designed and arrayed as reference spots.
  • HLA-DQB1 locus specific primers Table 3
  • purified DNA samples from the 13th International Histocompatibility Workshop (IHWS) cell bank
  • dried blood spot samples from The Environmental Determinants of Diabetes in the Young (TEDDY) study. All of the samples used had been genotyped (and some also sequenced) previously.
  • a prewash with 10 mM NaOH and boiling in MQ water for 10 min had to be done prior to PCR reaction to elute the DNA from the filter card.
  • the PCR amplification of the target strands can be performed with any amplification kit known in the art.
  • PCR reactions contained Taq98TM Hot Start polymerase 1x master mix (Lucigen), 0.5 ⁇ forward and 0.5 ⁇ reverse primers and the DNA sample.
  • the amount of starting material per reaction was 50 ng and in the case of blood spot elutions it was 1/20 of the total reaction volume.
  • the PCR cycling was done according to manufacturer ' s instructions.
  • the single stranded, fluorescently labelled DNA samples were hybridized on the oligonucleotide array as 1 :20 dilution in 6xSSC buffer with added 0.5 % Tween20 and 20 % formamide. Each sample was pipetted as two to four replicate wells. Hybridizations were performed in +37°C in 600 rpm for one hour. The plate was washed twice with TBS buffer + 0.05 % Tween20 and then dried. Fluorescent signals from array spots were measured by scanning the plate with LS400 microarray scanner (Tecan). The Cy5 signals were measured using 633 nm excitation and 670 nm emission wavelengths.
  • the basic idea in present multi-unit probes is that at least two binding units are joined by at least one linking unit, wherein the multi-unit probe hybridizes stably to the target nucleic acid in stringent conditions only when all binding units bind to their respective complementary regions.
  • the binding units are designed so that alone they do not form a stable hybrid with the target.
  • This basic idea of the current invention was demonstrated using closely related group of DQB1 * 03 alleles as an example. DQB1 * 03 alleles are highly homologues with no single discriminating SNPs.
  • Two target motifs A and B selected and designed as described above, were used to construct four different probe types; single short probes (A or B), mixed short probes (A+B), multi-unit probes (A-linker-B) and long probes (A-B with full sequence in between).
  • Multi- unit probes gave 3-7 times higher signals than mixed probes, separating the specific signals better from the unspecific or background signals, therefore bringing higher sensitivity and increased specificity to the assay.
  • Decreased cross-reactivity of the multi-unit probes towards unspecific target samples compared to mixed probes is further demonstrated in Figure 5.
  • Cross-reactivity between the samples remains below 25 % in all cases.
  • the increased sensitivity and specificity is most likely due to equal molar amounts and precise distance of the sequence units in the multi-unit probes, as compared to mixed probes.
  • T1 D type 1 diabetes
  • HLA region especially within the DRB1 -DQA1 -DQB1 loci.
  • Specific haplotypes and genotypes are responsible for high disease predisposing risk whereas others are protective in nature.
  • T1 D disease risk evaluation it is valuable to identify these two alleles in order to exclude those persons from follow-up research, as these persons will most unlikely get the disease.
  • the TripleGrip probe was designed to contain recognition elements around three polymorphic regions that in combination are known to separate * 0403 and * 0406 alleles from other * 04xx alleles. These include CAAC polymorphism at bases 27-28, T/G polymorphism at base 64 and ATC/GCT polymorphism at bases 1 14-1 16. The numbers represent base numbers in the PCR amplified antisense strand of DRB1 loci.
  • Binding units of the TripleGrip probe were constructed around the above mentioned polymorphic regions using the design strategy and criteria presented in the claims of this invention and in more detail in Example 1 .
  • the designed binding units for the * 0403/6 TripleGrip probe are presented in Figure 7 (polymorphic bases marked with arrows). The binding units are connected in the final probe sequence with a five thymidine oligomer, dT(5), linker. Amino- modified * 0403/6 TripleGrip probe was attached to 96-well bottoms as described in oligonucleotide array printing protocol in Example 1 . Also the PCR amplification of the target molecules and hybridizations were performed as described in Example 1 . Sequences for HLA-DRB1 * 04 specific primers are presented in Table 4. Table 4. HLA-DRB1*04 locus specific primers
  • the design strategy of the multi-grip probes is applicable also to targets where more than two distantly located recognition elements need to be connected into a single probe.
  • a unique combination of recognition elements allows specific identification of the target molecules without cross-reactivity from other closely related targets. Therefore the multi-grip probes offer superior functionality not reachable by other probe design means.

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Abstract

La présente invention porte sur une sonde à plusieurs motifs qui se lie à un acide nucléique cible grâce à au moins deux motifs de liaison complémentaires à des motifs cibles discontinus dans l'acide nucléique cible. De façon importante, les motifs de liaison sont conçus de façon à ce qu'une hybridation stable ait lieu dans des conditions de stringence seulement lorsque tous les motifs de liaison se lient à leurs motifs cibles respectifs. Les présentes sondes peuvent faire la différence entre des séquences d'acide nucléique hautement homologues et peuvent par conséquent être utilisées en typage HLA. De plus, l'invention porte sur des puces à ADN et sur un procédé de conception des présentes sondes.
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WO2017041084A3 (fr) * 2015-09-03 2017-04-27 Nanostring Technologies, Inc. Sondes multivalentes ayant une résolution de nucléotide simple
WO2021234172A1 (fr) * 2020-05-22 2021-11-25 University Of Ulster Test génétique pour prédire la réponse aux médicaments anti-tnf
EP3766992A4 (fr) * 2018-03-15 2021-12-01 Eiken Kagaku Kabushiki Kaisha Sonde oligonucléotidique pour détection de polymorphisme mononucléotidique, et procédé de différenciation de forme cis et de forme trans

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WO2017041084A3 (fr) * 2015-09-03 2017-04-27 Nanostring Technologies, Inc. Sondes multivalentes ayant une résolution de nucléotide simple
EP3766992A4 (fr) * 2018-03-15 2021-12-01 Eiken Kagaku Kabushiki Kaisha Sonde oligonucléotidique pour détection de polymorphisme mononucléotidique, et procédé de différenciation de forme cis et de forme trans
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WO2021234172A1 (fr) * 2020-05-22 2021-11-25 University Of Ulster Test génétique pour prédire la réponse aux médicaments anti-tnf

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