SE523727C2 - Method for selecting most sensitive nucleic acid probes for diagnostic purposes - Google Patents

Method for selecting most sensitive nucleic acid probes for diagnostic purposes

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Publication number
SE523727C2
SE523727C2 SE0103069A SE0103069A SE523727C2 SE 523727 C2 SE523727 C2 SE 523727C2 SE 0103069 A SE0103069 A SE 0103069A SE 0103069 A SE0103069 A SE 0103069A SE 523727 C2 SE523727 C2 SE 523727C2
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nucleic acid
hybridization
probes
probe
sid
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SE0103069A
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SE0103069D0 (en
SE0103069L (en
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Mikael Kubista
Nicke Svanvik
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Lightup Technologies Ab
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Publication of SE0103069L publication Critical patent/SE0103069L/en
Publication of SE523727C2 publication Critical patent/SE523727C2/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811Selection methods for production or design of target specific oligonucleotides or binding molecules
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/6818Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
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  • Analytical Chemistry (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Method for selection of hybridization probes (I), consisting of sequence recognition part (SID) and reporter group (RG) and able to bind to different parts of a target sequence (II), by providing at least 10, best at least 1000, different (I) on a solid matrix, then detecting signal from RG in presence and/or absence of hybridization to (II), is new. Independent claims are also included for the following: (a) preparation of (I), complementary to different parts of (II), by attaching oligoribonucleic acids or nucleic acid analogs to a solid matrix; and (b) a matrix to which (I) are attached.

Description

vlsan sara: 10 15 20 25 30 7523 727 u o II sun 2 För att påvisa en specifik mutation är urvalet mer begränsat, eftersom sondens sekvens måste överlappa den presumtiva mutationen, men det ñnns ändock flera alternativ. vlsan sara: 10 15 20 25 30 7523 727 u o II sun 2 To detect a specific mutation, the selection is more limited, as the sequence of the probe must overlap the presumptive mutation, but there are still alternatives.

I PCT/SE97/00953 föreslås att man välj er SID:s sekvens utifrån RG:s kända egenskaper, och för asymmetriska cyaninfargämnen föreslås SID:s vars tenninala baser är företrädesvis blandade pyrimidiner (-TT eller -CT). Denna strategi har flera begränsningar. Dels krävs detaljerade studier av fargämnets egenskaper som dessutom inte trivialt kan extrapoleras till sondemas egenskaper, p. g.a. skillnader i experimentella betingelser etc., dels uppfylls sekvenskraven i regel av ett obekvämt stort antal sekvenser ( 1/8 av alla sekvenser, t.ex. slutar med antingen -TT eller -CT). Slutligen tas ingen hänsyn till sekvensen intill MS.In PCT / SE97 / 00953 it is proposed to select the sequence of SID based on the known properties of RG, and for asymmetric cyanine dyes it is proposed SIDs whose tenninal bases are preferably mixed pyrimidines (-TT or -CT). This strategy has fl your limitations. On the one hand, detailed studies of the properties of the dye are required, which moreover cannot be trivially extrapolated to the properties of the probes, p. G.a. differences in experimental conditions, etc., and the sequence requirements are generally met by an uncomfortably large number of sequences (1/8 of all sequences, eg ending with either -TT or -CT). Finally, no account is taken of the sequence adjacent to the MS.

Föreliggande uppfinning löser dessa problem. Den utgör grundmässigt ett förfarande för att undersöka vilken av ett stort antal potentiella sonder för en viss nukleinsyra som har den lägsta bakgrundssignalen och vilken av dessa som erhåller starkast signal vid hybridisering.The present invention solves these problems. It is basically a method for examining which of a large number of potential probes for a particular nucleic acid has the lowest background signal and which of these receives the strongest signal during hybridization.

Skillnaden i bestämningar är den signalökning som erhålls med de olika sonderna.The difference in determinations is the signal increase obtained with the different probes.

Beskrivning av figurer Figur 1. Princip för homogen testning.Description of figures Figure 1. Principle of homogeneous testing.

Figur 2. Sond för homogen testning bestående av en sekvens-igenkännande del (SID) och en reportergrupp (RG).Figure 2. Homogeneous testing probe consisting of a sequence recognition part (SID) and a reporter group (RG).

Figur 3. Princip som visar hur den ur känslighetshänseende lämpligaste sonden för viss nukleinsyra kan identifieras.Figure 3. Principle showing how the most sensitive probe for a particular nucleic acid can be identified.

A: Exempel på sonder som alla känner igen samma nukleinsyra.A: Examples of probes that all recognize the same nucleic acid.

B: Dessa sonder syntetiseras på en fast matris.B: These probes are synthesized on a solid matrix.

C: Fluorescensen hos sonder mäts i såväl fiitt som hybridiserat tillstånd. De som ger upphov till störst signalökning är de lämpligaste.C: The fluorescence of probes is measured in both fi single and hybridized state. Those that give rise to the largest signal increase are the most suitable.

Figur 4 visar olika sonder och deras olika fluoroscensintensitet före hybridisering Figur 5 illustrerar en sondkonstruktion beskriven i utföringsexempel tillsammans med sekvensmodifiering för de olika sondema, vars intensitet visas i Figur 4.Figure 4 shows different probes and their different fluorescence intensities before hybridization Figure 5 illustrates a probe construction described in exemplary embodiments together with sequence modification for the different probes, the intensity of which is shown in Figure 4.

Innan Ina-n 10 15 20 25 30 säs 727 Beskrivning av uppfinningen Deoxyribonukleinsyror och flera nukleinsyraanalo ger, såsom t.ex. peptid nukleinsyror (PNA), syntetiseras vanligen med fastfas syntes, vilket bl. a. tillåter syntes av stort antal fragment med olika sekvenser och även olika längder på en matris (Khrapko et al., FEBS Lett. 256, 118. 1989; Southern, E., et al., Genomics 13, 1008, 1992; Caviani-Pease, et al., Proc. Natt. Acad. Sci., 91, 5022, 1994; Weiler et al., Nucl. Acids Res. 25, 2792, 1997).Description of the Invention Deoxyribonucleic acids and several nucleic acid analogs, such as e.g. peptide nucleic acids (PNA), are usually synthesized by solid phase synthesis, which i.a. a. allows the synthesis of large numbers of fragments with different sequences and also different lengths of a matrix (Khrapko et al., FEBS Lett. 256, 118. 1989; Southern, E., et al., Genomics 13, 1008, 1992; Caviani -Pease, et al., Proc. Natt. Acad. Sci., 91, 5022, 1994; Weiler et al., Nucl. Acids Res. 25, 2792, 1997).

Fragmenten på denna matris kan hybridiseras med märkt nukleinsyra , t.ex. med fluoreseenta eller radioaktiva grupper, och graden av hybridisering kan bestämmas från nukleinsyrans signal. Denna teknologi har många applikationer, och kallas ofta för DNA-chip teknologi.The fragments on this matrix can be hybridized with labeled nucleic acid, e.g. with or oresent or radioactive groups, and the degree of hybridization can be determined from the nucleic acid signal. This technology has many applications, and is often referred to as DNA chip technology.

I föreliggande uppfinning utnyttjas DNA-chip teknologin för att avgöra vilka av ett stort antal sonder, som alla, med tillräcklig specificitet, känner igen viss nukleinsyra, som lämpar sig bäst för nukleinsyrahybridisering, och då företrädesvis i homogen lösning. Sonder, dvs oligodeoxyribonukleinsyror eller nukleinsyraanaloger utrustade med reportergrupper (RG), som alla är tillräckligt komplementära och tillräckligt specifika för viss nukleinsyra, syntetiseras på en matris (Figur 3). Signalen, företrädesvis fluorescensen, som de ger upphov till, dvs bakgrundssignalen registreras. Därefter tillsätts viss nukleinsyra och fluorescensen, denna gång från hybridiserade sonder, registreras. Skillnaden i signal i dessa mätningar är den signalökning som erhålles med de olika sonderna. De, som uppvisar störst signalskillnad, är de, som ur känslighetshänseende, är lämpligast.In the present invention, DNA chip technology is used to determine which of a large number of probes, all of which, with sufficient specificity, recognize certain nucleic acid, which is best suited for nucleic acid hybridization, and then preferably in homogeneous solution. Probes, ie oligodeoxyribonucleic acids or nucleic acid analogues equipped with reporter groups (RGs), all of which are sufficiently complementary and specific for a particular nucleic acid, are synthesized on a matrix (Figure 3). The signal, preferably the uorescence, which they give rise to, ie the background signal is registered. Then some nucleic acid is added and the orescence, this time from hybridized probes, is recorded. The difference in signal in these measurements is the signal increase obtained with the different probes. The ones that show the greatest signal difference are the ones that are most suitable in terms of sensitivity.

Olika matrismaterial kan användas, såsom cellulosasubstrat, metallsubstrat och polymersubstrat. Vid användning av metallsubstrat belägges oftast metallen först med ett aminosyraskikt för att underlätta vidhäñning. Vid användning av cellulosasubstrat anbringas ofta en första nukleotid på substratet, varefter ytterligare nukleotider syntetiseras ovanpå den först anbringade tills man erhållit önskad, lämplig nukleotidsekvens.Various matrix materials can be used, such as cellulose substrates, metal substrates and polymer substrates. When using metal substrates, the metal is usually first coated with an amino acid layer to facilitate adhesion. When using cellulose substrates, a first nucleotide is often applied to the substrate, after which additional nucleotides are synthesized on top of the first applied until the desired, appropriate nucleotide sequence is obtained.

Föreliggande fasta sonder kan användas för analys av nukleinsyra/or i form av mRNA, DNA, PNA, PNA-PNA komplex, eller DNA-PNA komplex.The present solid probes can be used for the analysis of nucleic acid (s) in the form of mRNA, DNA, PNA, PNA-PNA complex, or DNA-PNA complex.

Signalförändring vid hybridisering kan ske antingen genom att sonden ändrar sina egenskaper, signalförändringen icke-hybridiserat tillstånd visavi hybridiserat tillstånd, eller att man hybridiserar till en märkt nukleinsyra med en annan reportergrupp, RG°, varvid -uann Iløan 10 15 20 25 30 525'727 4 signalfórändringen erhålles då RG-gruppen i sonden och RGïgruppen i målDNA:t kommer i varandras närhetRG och RG' kan vara lika eller olika. Vid system innehållande pyren ändras fluorescens egenskaperna markant när två stycken pyrener kommer i kontakt, varvid erhålles excimer fluoroscens. Exempel på två olika RG, RG' är energiöverfóringspar (energy transfer pair) såsom fluoroscein/tetrametylrhodamin eller fluorofor/utsläckarepar (fluorophore/quencher pair).Signal change during hybridization can occur either by the probe changing its properties, the signal change non-hybridized state versus hybridized state, or by hybridizing to a labeled nucleic acid with another reporter group, RG °, wherein -uann Iløan 10 15 20 25 30 525'727 The signal change is obtained when the RG group in the probe and the RG1 group in the target DNA come in close proximity. RG and RG 'may be the same or different. In systems containing pyrene, the fl uorescence properties change markedly when two pieces of pyrene come into contact, whereby excimer fl uoroscence is obtained. Examples of two different RG, RG 'are energy transfer pairs such as fl uoroscein / tetramethylrhodamine or fl uorophore / quencher pair.

Uppfinningen kommer att beskrivas nedan med hänvisning till ett exempel, som illustrerar uppfinningen utan att denna dock är begränsad härtill.The invention will be described below with reference to an example which illustrates the invention without, however, being limited thereto.

Exempel Femton 10-baser långa PNA-tiazolorange sonder, komplementära till olika segment för sekvensen GTCAGATGAGGAAGAGGCTATTGT, och en sond, komplementär i parallell orientering till den centrala polypurinregionen, syntetiserades på ett Perseptive-PP-NHZ- membran (åtskilda från membranet med PEG-500-Glu-Lys-kapronsyra länk) med användning av en ABIMED ASP 222 Automated SPOT Robot (Weiler, J. et al., Nucleic Acids Res., 25, 2792, (1997), Figure 1). PNA monomerema anbringades som beskrives av Weiler, J. et al., supra. I sista steget aktiverades tiazolorange-fargen substituerad med en karboxylsyraalkyl-lärik och reagerades på samma sätt som Fmoc-PNA monomererna. Eñer syntes eliminerades från PNA oligomerema sidokedjeskyddsgruppema genom behandling med 90% TFA! 5% vatten/ 5% trietylsilan under 1 timme (TFA = trifluorättikssyra).Example Fifteen 10-base long PNA-thiazolorange probes, complementary to different segments of the sequence GTCAGATGAGGAAGAGGCTCTTGT, and a probe, complementary in parallel orientation to the central polypurine region, were synthesized on a Perseptive-PP-NHZ membrane (separated from the P-500 membrane by 500 -Glu-Lys-caproic acid link) using an ABIMED ASP 222 Automated SPOT Robot (Weiler, J. et al., Nucleic Acids Res., 25, 2792, (1997), Figure 1). The PNA monomers were applied as described by Weiler, J. et al., Supra. In the final step, the thiazole orange dye substituted with a carboxylic acid alkyl larch was activated and reacted in the same manner as the Fmoc-PNA monomers. One synthesis was eliminated from the PNA oligomers side chain protecting groups by treatment with 90% TFA! 5% water / 5% triethylsilane for 1 hour (TFA = triacetic acid).

Membranet fuktades sedan i en 10 mM boratbuffert vid pH 8,5 innehållande en tillsats av 100 mM NaCl under 2 timmar och belystes med en standard UV-lampa med Ämax = 312 nm, och fotograferades med en CCD kamera (Figur 4). Bakgrundsfluoroscensen för sondema har uttryckts relativt sond 16 (CCTCTTCCTC-TO), vilken uppvisar den svagaste intensiteten fore hybridisering, och vilken därmed förväntas ge den största signalförändringen efter hybridisering..The membrane was then moistened in a 10 mM borate buffer at pH 8.5 containing an addition of 100 mM NaCl for 2 hours and illuminated with a standard UV lamp with Ämax = 312 nm, and photographed with a CCD camera (Figure 4). The background fluorescence of the probes has been expressed relative to probe 16 (CCTCTTCCTC-TO), which shows the weakest intensity before hybridization, and which is thus expected to give the largest signal change after hybridization.

Claims (5)

10 15 20 25 30 : e < = .e f i i I 5 . ._ v vw o p e f n- - 1 n n na a oo o u | . ' ' I 0 Ir ro :men :se PATENTKRAV Ö10 15 20 25 30: e <= .e f i i I 5. ._ v vw o p e f n- - 1 n n na a oo o u | . '' I 0 Ir ro: men: se PATENTKRAV Ö 1. Selektering av sonder för detektion av nukleinsyra medelst hybridisering, vilka sonder består av en sekvensigenkännande del, SID, och en reportergrupp, RG, där sondens påvisbara egenskaper ändras vid hybridisering till en nukleinsyra, varvid SID's sekvens valts ut genom att jämföra RG signalen i fritt och hybridiserat tillstånd hos åtminstone 10, företrädesvis åtminstone 100 och, mera företrädesvis åtminstone 1000 sonder, som hybridiserar till olika delar av viss nukleinsyra, varvid sonderna är syntetiserade till en fast matris och varvid deras signal bestämmesi frånvaro av viss nukleinsyra, och deras signal bestämmes i närvaro av viss nukleinsyra.Selecting probes for nucleic acid detection by hybridization, which probes consist of a sequence recognition moiety, SID, and a reporter group, RG, wherein the detectable properties of the probe change upon hybridization to a nucleic acid, the sequence of SID being selected by comparing the RG signal in free and hybridized state of at least 10, preferably at least 100 and, more preferably at least 1000 probes, which hybridize to different parts of certain nucleic acid, the probes being synthesized to a solid matrix and their signal being determined in the absence of certain nucleic acid, and their signal being determined in the presence of certain nucleic acid. 2. Selektering enligt krav 1, där hybridisering sker i homogen lösning.Selection according to claim 1, wherein hybridization takes place in homogeneous solution. 3. Selektering enligt ett eller flera av kraven 1-2, kännetecknat av, att sonden, omfattande SID och RG, är utformad att ändra sina egenskaper vid hybridisering till en målsekvens.Selection according to one or more of Claims 1 to 2, characterized in that the probe, comprising SID and RG, is designed to change its properties during hybridization to a target sequence. 4. Selektering enligt ett eller flera av kraven 1-3, kännetecknat av, att sonden, omfattande SID och RG, är utformad att ge en signalförändring genom sin RG vid hybridisering till en målsekvens.Selection according to one or more of claims 1-3, characterized in that the probe, comprising SID and RG, is designed to give a signal change through its RG upon hybridization to a target sequence. 5. Selektering enligt ett eller flera av kraven 1-4, I kännetecknat av, att sonden omfattande SID och RG är utformad att hybridisera till en märkt målsekvens innehållande en reportergrupp RG' som är lika med eller skild från RG i sonden, varvid en signalförändring är anordnad att uppstå då RG och RG' kommer i varandras närhet vid hybridisering.Selection according to one or more of claims 1-4, characterized in that the probe comprising SID and RG is designed to hybridize to a labeled target sequence containing a reporter group RG 'which is equal to or different from RG in the probe, wherein a signal change are arranged to occur when RG and RG 'come in close proximity to each other during hybridization.
SE0103069A 2001-09-13 2001-09-13 Method for selecting most sensitive nucleic acid probes for diagnostic purposes SE523727C2 (en)

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