DE102014210092A1 - Apparatus and method for detecting and quantifying single-stranded target nucleic acids - Google Patents

Abstract

The invention relates to an apparatus and a method for the detection and quantification of single-stranded target nucleic acids. The device comprises at least one solid support on which at least one double-stranded capture oligonucleotide and at least one double-stranded reporter oligonucleotide are immobilized. The capture oligonucleotide comprises at least a first oligonucleotide sequence which is partially complementary to a target nucleic acid and the reporter oligonucleotide comprises at least a second oligonucleotide sequence which is partially complementary to the target nucleic acids. Furthermore, the device comprises a readout unit for reading out a label of the reporter oligonucleotide on the carrier.

Description

The invention relates to an apparatus and a method for the detection and quantification of single-stranded target nucleic acids.

The expression of genes in cells is strictly controlled and regulated by a variety of molecular processes. In addition to the complex regulation of transcription of genes, mechanisms at the post-transcriptional level, that is, at the level of mRNA, also play an important role. In particular, short, noncoding nucleic acid molecules can interfere with gene expression by specifically attaching to complementary sequences of mRNA molecules and thereby altering the translation rate of the mRNA into the corresponding protein. The short nucleic acid molecules are often so-called microRNAs (short "miRNAs"), which have in recent years increasingly aroused the interest of molecular biological and medical research. It has been shown that selected miRNAs are directly related to the development of specific cancers such as breast cancer, lung cancer or leukemia. The cancer cells have an increased or decreased number of specific miRNAs. Even with immunological diseases, such as rheumatism, certain miRNAs occur in altered concentrations. It is assumed that more than 1000 different miRNA molecules are found in humans alone.

Due to the growing importance and variety of miRNAs, methods for their specific identification and quantification are needed. Various methods for the detection of miRNAs are known, for example the sequencing of the miRNAs or the amplification of miRNAs by means of real-time PCR.

The US 6,322,971 describes a hybridization-based method for detecting a nucleic acid. In this case, the nucleic acid to be detected is covalently linked to a second, labeled nucleic acid after the two nucleic acids have been attached to an immobilized counterstrand oligonucleotide and missing nucleotides between the nucleic acid to be detected and the labeled nucleic acid have been filled in by a DNA polymerase. Unbound labeled nucleic acids are removed by raising the temperature.

The US 6,344,316 describes a method for the detection of nucleic acids, in which an immobilized capture oligonucleotide is covalently linked to a second, labeled oligonucleotide after the oligonucleotides have attached to the nucleic acid to be detected as a complementary strand. After removal of the nucleic acid to be detected by washing at high temperature, the covalently bound label remains.

From the prior art, various problems arise. For example, the specificity and sensitivity of miRNA detection are inadequate. Among other things, in hybridization-based methods, the low specificity is that no complete hybridization of the miRNA is required for the identification process. Because of the low sensitivity of the methods, the miRNAs often need to be amplified prior to their detection. This can lead to the incorporation of defective nucleotides, so that the specificity of miRNA detection is further limited. The procedures must also be performed at low temperatures because the short length of miRNA hybrids is associated with low melting temperatures. Thus, the miRNA detection is neither particularly robust nor quantitatively evaluable. In addition, many methods require pre-analytical labeling of miRNA, which entails the risk of inaccurate results and artifacts.

The invention is therefore based on the object to provide an apparatus and a method for the detection of single-stranded nucleic acids, which solves the problems mentioned.

The device according to the invention for detecting at least one single-stranded target nucleic acid in a sample comprises at least one solid support on which at least one double-stranded capture oligonucleotide is immobilized. The capture oligonucleotide comprises a first capture oligonucleotide partial strand and a second capture oligonucleotide partial strand. The first end of the first capture oligonucleotide substrand is tightly attached to the support. The second capture oligonucleotide substrand has a first capture oligonucleotide overhang on the side away from the support.

Furthermore, the device according to the invention comprises a double-stranded reporter oligonucleotide having a first and a second reporter oligonucleotide partial strand. A first end of the second reporter oligonucleotide substrand comprises a first single-stranded reporter oligonucleotide overhang.

The first capture oligonucleotide overhang comprises at least a first oligonucleotide sequence that is complementary to a first target nucleic acid segment. The first reporter oligonucleotide overhang comprises at least a second oligonucleotide sequence which complements to a second target nucleic acid portion is. The first capture oligonucleotide overhang thus hybridizes to the first target nucleic acid portion and the first reporter oligonucleotide overhang hybridizes to the second target nucleic acid portion.

Furthermore, the device according to the invention comprises a read-out unit for reading out a label of the first reporter oligonucleotide partial strand on the carrier.

The method according to the invention for the detection and quantification of at least one single-stranded target nucleic acid comprises a plurality of steps: providing at least one solid support on which at least one double-stranded capture oligonucleotide is immobilized. The capture oligonucleotide comprises a first capture oligonucleotide partial strand and a second capture oligonucleotide partial strand. The first end of the first capture oligonucleotide substrand is attached to the support. The second capture oligonucleotide substrand has a first capture oligonucleotide overhang on the side away from the support.

A further step is contacting the support with at least one single-stranded target nucleic acid and at least one double-stranded reporter oligonucleotide in a first hybridization condition, wherein the reporter oligonucleotide comprises a first and a second reporter oligonucleotide sub-strand. A first end of the second reporter oligonucleotide substrand comprises a first single-stranded reporter oligonucleotide overhang.

The first capture oligonucleotide overhang comprises at least a first oligonucleotide sequence that is complementary to a first target nucleic acid portion. Furthermore, the first reporter oligonucleotide overhang comprises at least one second oligonucleotide sequence that is complementary to a second target nucleic acid portion, such that the first capture oligonucleotide overhang with the first target nucleic acid portion and the first reporter oligonucleotide Overhang hybridize with the second target nucleic acid section.

The method further comprises the step of ligating the target nucleic acid to the first capture oligonucleotide substrand and to the first reporter oligonucleotide subset by means of a ligase. Furthermore, it comprises a first washing of the carrier under stringent washing conditions such that the carrier is cleaned of unligated nucleic acids. Another step is reading out a label of the first reporter oligonucleotide partial strand on the support.

The term target nucleic acid refers to a single-stranded nucleic acid having a length of in particular about 17 to about 50 nucleotides. The nucleic acid may be an RNA or a DNA. In particular, the nucleic acids are naturally occurring nucleic acids as well as chemically produced or recombinantly produced nucleic acids.

The term "hybridize" refers to the attachment of a single-stranded RNA or DNA to an at least partially complementary single-stranded RNA or DNA to form hydrogen bonds between the respective complementary bases, so that base pairings form in the mutually complementary section between the two nucleic acid molecules.

The term "hybridization condition" refers to at least one parameter or a combination of parameters in which at least one of the hybridization steps of the method is performed. The hybridization condition is a condition in which the capture oligonucleotide, the target nucleic acid, and / or the reporter oligonucleotide anneal to the respective complementary portions of the opposite strand oligonucleotide to form base pairings. The parameters preferably include a temperature, a salt concentration, an ionic strength, a pH and / or an addition of reagents such as formamide.

By means of the device according to the invention and the method according to the invention, the specificity of the analysis of the target nucleic acid is markedly increased. The target nucleic acid is completely bound to the reporter and capture oligonucleotide. Thus, each base is analyzed without omitting defined portions of the target nucleic acid. This advantageously improves the discrimination of target nucleic acids which differ in marginal nucleotides.

Furthermore, the sensitivity is advantageously increased. After the step of hybridizing the target nucleic acid to the double strand of the capture oligonucleotide, or the reporter oligonucleotide, and then ligation of the target nucleic acid with the capture oligonucleotide and the reporter oligonucleotide, the target nucleic acid is both solid to the carrier, as well as firmly attached to the label. In the washing step, therefore, only unbound or weakly bound reporter oligonucleotides or non-target nucleic acids are very selectively removed from the carrier. Advantageously, this significantly reduces false-positive signals and thus increases the sensitivity. By increasing the sensitivity, amplification of the target nucleic acid prior to analysis can advantageously be avoided.

The method according to the invention is a quantitative measuring method which advantageously requires very short test times for detecting the target nucleic acid.

In an advantageous development and embodiment of the invention, the first capture oligonucleotide overhang and / or the first reporter oligonucleotide overhang comprises eight to 25 bases. The length of each overhang depends on the length of the target nucleic acid. The target nucleic acid or its complementary sequence is split between the reporter and capture oligonucleotide overhang. This partitioning is typically done so that each nucleotide of the target nucleic acid can be hybridized to a reporter or capture overhang. However, it is also conceivable that a nucleotide sequence or a single base of the target nucleic acid is bound neither to the reporter oligonucleotide nor to the capture oligonucleotide. This is particularly advantageous for the detection of single nucleotide polymorphisms.

In a further advantageous embodiment and development of the invention, the first end of the first capture oligonucleotide partial strand comprises six thymine bases. The capture oligonucleotide is advantageously bound to the support with at least one thymine base.

In a further advantageous embodiment and development of the invention, the first reporter oligonucleotide partial strand on the label. Particularly preferably, the first reporter oligonucleotide partial strand has the label at its distal end to the carrier or to the target nucleic acid. The label is then tightly bound to the support during ligation via the target nucleic acid and the first capture oligonucleotide partial strand.

In a further advantageous embodiment and development of the invention, the ligase is a T4 DNA ligase. T4 DNA ligase is an enzyme that catalyzes the ATP-dependent ligation of two single-stranded RNA or DNA molecules. A covalent bond of the 3'-hydroxyl end of a first nucleic acid molecule to the 5'-phosphoryl end of a second nucleic acid molecule is thereby produced. In particular, the T4 DNA ligase can ligate two DNA molecules with each other, two RNA molecules with each other and a DNA molecule with an RNA molecule. This is necessary because the repoter and capture oligonucleotide typically comprise DNA nucleotides and the target nucleic acid typically comprises RNA nucleotides. However, T4 DNA ligase only ligates the 5 'end of a DNA with a 3' end of an RNA. It does not ligate a 3 'end of the DNA with a 5' end of the RNA. Therefore, in a further advantageous embodiment and development of the invention, the 3 'end of the DNA is synthesized with at least one, preferably four, RNA nucleotides as a chimera to allow ligation. This concerns in particular the first capture oligonucleotide partial strand. This is bound with its 5 'end to the carrier. The 3 'end of the first capture oligonucleotide substrand is here ligated to the 5' end of the target nucleic acid. However, it is also conceivable that the orientation of the partial strands is bound in opposite directions on the carrier.

Furthermore, to ligate DNA and RNA with T4 DNA ligase, the 5 'end of the DNA and the RNA must be phosphorylated. The target nucleic acid is in particular a miRNA. This is already naturally phosphorylated in the body. The reporter or capture oligonucleotide must be synthesized according to phosphorylated.

In general, the following prerequisites for ligation must be given for T4 DNA ligase: The 5 'ends to be ligated must be present in phosphorylated form. In the case where the target nucleic acid is an RNA, the 3 'ends of the reporter or capture oligonucleotide to be ligated must comprise RNA nucleotides. This can be designed as an RNA strand or as a chimera, the chimera being fundamentally more stable.

In a further advantageous embodiment and development of the invention, a second washing of the carrier takes place under mild washing conditions prior to ligation. Under mild washing conditions, the salt concentration is increased over the harsh wash conditions and / or the temperature is lowered. In harsh wash conditions, however, washed with low salt concentrations and / or high temperatures.

In a further advantageous embodiment and development of the invention, at least two different target nucleic acids, preferably 3 to 130 different nucleic acids, are read out simultaneously. Advantageously, the carrier is then designed as a chip.

In a further advantageous embodiment and development of the invention, the ligation is carried out at 37 ° C.

In a further advantageous embodiment and development of the invention, the readout of the label is carried out by means of a "branched DNA" test and the reporter oligonucleotide is preferably a "preamplifier." The "branched DNA" test ("branched DNA", in German: branched DNA), also known as "branched DNA Assay", is a signal amplification assay for amplifying the reporter signal, using a so-called "preamplifier" (in German: preamplifier). used, which hybridizes directly or indirectly to the target nucleic acid to be detected. In subsequent steps, so-called "amplifiers" (in German: amplifiers) are hybridized to the "preamplifier". "Preamplifier" and "Amplifier" are short single-stranded oligodeoxyribonucleotides. The "amplifiers" are branched oligodeoxyribonucleotides that bind reporter molecules. Signal amplification is achieved by attaching multiple "amplifiers" to a preamplifier and each "amplifier" typically binds multiple reporter molecules.

By branching the "amplifiers", a high density of "amplifiers" on the "preamplifier" can be achieved, which leads to a high sensitivity of the "branched DNA" test Depending on the labeling used, in particular enzyme labeling or bDNA assay, concentrations of the target nucleic acid of less than 30 μM can be detected in particular when using an enzyme at test times of less than 30 minutes Test are detectable for test times less than 45 minutes concentrations of less than 100fM.

The invention will be further explained below with reference to the drawings.

1 schematically shows the steps of the method for the analysis of an RNA at the times t1 to t5.

2 schematically shows a carrier with two capture oligonucleotides at time t1.

3 schematically shows a carrier with two capture oligonucleotides, two reporter oligonucleotides and two miRNAs at time t2.

4 schematically shows a support with two capture oligonucleotides and a reporter oligonucleotide at time t2 '.

5 schematically shows a support with two capture oligonucleotides and two reporter oligonucleotides, two miRNAs and the T4 DNA ligase at time t3.

6 Figure 16 shows schematically the miRNA with a capture oligonucleotide single strand and with a reporter oligonucleotide single strand ligated at time t4.

7 schematically shows catcher oligonucleotide single strand after harsh wash conditions without miRNA presence at time t4 '.

1 schematically shows the steps of the method for detecting and quantifying a miRNA. In this example, the target nucleic acid is a miRNA named miR-16 5 , MiR-16 5 is a miRNA that can reduce the expression of the anti-apoptotic protein Bcl-2 in lymphocytes. The method can be exemplified in five steps in the presence of miRNA 5 subdivide (steps t1, t2, t3, t4 and t5). In the absence of a target nucleic acid 5 For example, in this example, the method includes steps t1, t2 ', t4' and t5 '.

In step t1, the provision of a carrier takes place 1 with double-stranded capture oligonucleotides immobilized thereon 2 ( 2 ). Here, the first capture oligonucleotide substrand is 14 with a first end of a second single-stranded capture oligonucleotide overhang 3 firmly attached to the carrier via a spacer 1 bound, ie immobilized. In particular, the spacer comprises six thymine bases. Alternatively, the first capture oligonucleotide substrand 14 with the first end bound to the support, with no oligonucleotide overhang present.

The first single-stranded capture oligonucleotide overhang 4 comprises an oligonucleotide sequence which results in the target nucleic acid miR-16 5 is partially complementary.

In the second exemplary method step at the time t2 or t2 '( 3 and 4 ) now becomes the reporter oligonucleotide 6 added to the system and the biological sample via the carrier 1 guided.

In the event that the biological sample contains the target nucleic acid miR-16 5 includes, this step is in 3 shown. In 3 is shown schematically that the capture oligonucleotide 2 with its first capture oligonucleotide overhang 4 with a first section of the miR16 5 hybridized. Furthermore, it can be seen that a second portion of miR-16 attached to a second reporter oligonucleotide overhang 8th , which is complementary to the second portion of miR-16, hybridizes. The reporter oligonucleotide 6 comprises at a second reporter oligonucleotide overhang 9 a mark 7 , Alternatively, the two reporter oligonucleotide sub-strands 16 . 17 seen from the wearer distal end have no overhang, but be about the same length.

In this example, the single-stranded portions of the capture oligonucleotide are 4 and the reporter oligonucleotide 9 in such a way complementary, that no free sequence section in the center of the miRNA 5 remains free. Furthermore, the reporter oligonucleotide partial strand without marking comprises the a section of the target nucleic acid complementary section.

In the event that the biological sample does not have a target nucleic acid miR-16 5 , the step is at time t2 'in FIG 4 shown schematically. Even without the presence of a target nucleic acid 5 can be between reporter oligonucleotide 6 and capture oligonucleotide 2 weak and unspecific bonds arise.

In a next process step at time t3 ( 5 ) Ligase is now added to the system. This example is a T4 DNA ligase 13 , This ligase connects the first capture oligonucleotide substrand 14 at the ligation site 10 with the miRNA 5 and the first reporter oligonucleotide substrand 16 , These compounds are covalent bonds. This means that the first capture oligonucleotide substrand 14 , the miRNA 5 and the first reporter oligonucleotide substrand 16 are firmly connected. For the ligation of single strands 14 . 5 . 16 it is necessary that these border directly on each other. This is ensured by the fact that the two overhangs 4 . 9 the miRNA 5 bind completely complementary.

In the event that no target nucleic acid 5 is present in the biological sample, the T4 DNA ligase 13 the ends of the capture oligonucleotide 2 and the reporter oligonucleotide 6 not with the target nucleic acid 5 ligate.

The carrier 1 is then washed at a temperature of 55 ° C for 10 minutes with a salt-containing buffer solution. This will, in the presence of the miR-16 5 , the base pairings of the second capture oligonucleotide substrand 15 and the second reporter oligonucleotide substrand 17 melted and the dissolved single-stranded oligonucleotides 15 . 17 removed with the washing. This washing step is carried out with a low salt buffer.

Is not miR-16 5 present, dissolves during washing of the vehicle 1 at 55 ° C unspecifically bound reporter oligonucleotide 6 from the capture oligonucleotide 2 from. The reporter oligonucleotide 6 thus becomes the carrier 1 removed, leaving no mark 7 on the carrier 1 remains. Consequently, when reading the mark 7 no signal measured. 7 shows the capture oligonucleotide 2 after this washing process.

Also in the event that a miRNA is present which is not complementary to the capture oligonucleotide 2 or reporter oligonucleotide 6 is, becomes the reporter oligonucleotide 6 with the mark 7 washed away as there is no covalent bond after ligation. Thus, no signal is generated. This ensures a high specificity of the method for the respective target miRNA.

Subsequently, the carrier 1 brought to a readout temperature of 37 ° C at the mark 7 on the carrier 1 is evaluated. As a marker 7 becomes an enzyme to the reporter oligonucleotide 6 coupled. In this example, the enzyme is already bound to the reporter oligonucleotide as a label at the beginning of the detection procedure. Alternatively, the coupling of the enzyme in a further process step can be bound to the reporter oligonucleotide.

To read a signal, a substrate is placed on the carrier 1 given, which is converted by the enzyme. The product of this reaction is measured electrochemically by redox cycling on two electrodes. As the reporter enzyme, an esterase or an alkaline phosphatase can be used. Alternatively to a simple enzyme coupling to the reporter oligonucleotide 6 , signal amplification can also be performed using a branched DNA test. In this example, the "preamplifier" of the bDNA assay forms the subunit of the reporter, which is ligated to the target nucleic acid.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6322971 [0004]
• US 6344316 [0005]

Claims (12)

Contraption ( 20 ) for the detection of at least one single-stranded target nucleic acid ( 5 ) in a sample comprising: - at least one solid support ( 1 ) on which at least one double-stranded capture oligonucleotide ( 2 ) is immobilized, wherein the capture oligonucleotide ( 2 ) a first capture oligonucleotide substrand ( 14 ) and a second capture oligonucleotide subset ( 15 ), wherein a first end of the first capture oligonucleotide substrand ( 14 ) is firmly attached to the support and wherein the second capture oligonucleotide substrand ( 15 ) a first capture oligonucleotide overhang ( 4 ) at the of the carrier ( 1 ), - at least one double-stranded reporter oligonucleotide ( 6 ) with a first and a second reporter oligonucleotide substrand ( 16 . 17 ), wherein a first end of the second reporter oligonucleotide substrand ( 17 ) a first single-stranded reporter oligonucleotide overhang ( 9 ) and wherein the first capture oligonucleotide overhang ( 4 ) comprises at least a first oligonucleotide sequence which is complementary to a first target nucleic acid portion, and wherein the first reporter oligonucleotide overhang ( 9 ) comprises at least one second oligonucleotide sequence complementary to a second target nucleic acid portion to form the first capture oligonucleotide substrand ( 4 ) with the first target nucleic acid portion and the first reporter oligonucleotide overhang ( 9 ) to hybridize with the second target nucleic acid portion; A readout unit for reading a mark ( 7 ) of the first reporter oligonucleotide substrand ( 16 ) on the support ( 1 ). The device of claim 1, wherein the first capture oligonucleotide overhang ( 4 ) and / or the first reporter oligonucleotide overhang ( 9 ) comprise eight to 25 bases. Device according to one of claims 1 or 2, wherein the first end of the first capture oligonucleotide subunit ( 14 ) comprises six thymine bases. Device according to one of claims 1 to 3, wherein the first capture oligonucleotide partial strand is formed as a chimera with at least one, in particular with four, RNA nucleotides at its 3 'end. Method for detecting and quantifying at least one single-stranded target nucleic acid ( 5 ) comprising the steps of: a) providing at least one solid support ( 1 ) on the at least one double-stranded capture oligonucleotide ( 2 ) is immobilized, wherein the capture oligonucleotide ( 2 ) a first capture oligonucleotide substrand ( 14 ) and a second capture oligonucleotide subset ( 15 ), wherein a first end of the first capture oligonucleotide substrand ( 14 ) is firmly attached to the support and wherein the second capture oligonucleotide substrand ( 15 ) a first capture oligonucleotide overhang ( 4 ) at the of the carrier ( 1 ) facing away from the side, b) contacting the carrier ( 1 ) with at least one single-stranded target nucleic acid ( 5 ) and at least one double-stranded reporter oligonucleotide ( 6 ) at a first hybridization condition, - wherein the reporter oligonucleotide ( 6 ) a first and a second reporter oligonucleotide substrand ( 16 . 17 ), wherein a first end of the second reporter oligonucleotide substrand ( 17 ) a first single-stranded reporter oligonucleotide overhang ( 9 ) and wherein the first capture oligonucleotide overhang ( 4 ) comprises at least a first oligonucleotide sequence complementary to a first target nucleic acid portion, and wherein the first reporter oligonucleotide overhang ( 9 ) comprises at least one second oligonucleotide sequence complementary to a second target nucleic acid portion such that the first capture oligonucleotide overhang ( 4 ) with the first target nucleic acid portion and the first reporter oligonucleotide overhang ( 9 ) hybridize to the second target nucleic acid portion; c) Ligation of the target nucleic acid ( 5 ) to the first capture oligonucleotide substrand ( 14 ) and to the first reporter oligonucleotide substrand ( 16 ) by means of a ligase ( 13 ); d) First washing of the carrier ( 1 ) under stringent washing conditions such that the carrier ( 1 ) is purified from unligated nucleic acids; e) reading a mark ( 7 ) of the first reporter oligonucleotide substrand ( 16 ) on the support ( 1 ). Method according to claim 5 with an RNA as target nucleic acid. Method according to claim 6 with a miRNA ( 5 ) as RNA. Method according to one of claims 5 to 7 with a T4 DNA ligase ( 13 ) as ligase. Process according to any one of claims 5 to 8, wherein prior to ligation a second washing of the support ( 1 ) under mild washing conditions. Method according to one of claims 5 to 9, wherein at least two different target nucleic acids, preferably three to 130 different nucleic acids are read out simultaneously. The method of any of claims 5 to 10, wherein the ligation is performed at 37 ° C. Method according to one of claims 5 to 11, wherein the reading of the mark ( 7 ) by means of a "branched DNA" test and the reporter oligonucleotide ( 6 ) is preferably a "preamplifier".

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