WO1994012518A2

WO1994012518A2 - Reagent for coupling various substances to nucleic acids, and a method of preparing the reagent

Info

Publication number: WO1994012518A2
Application number: PCT/DE1993/001101
Authority: WO
Inventors: Gabor Igloi
Original assignee: Gabor Igloi
Priority date: 1992-11-25
Filing date: 1993-11-19
Publication date: 1994-06-09
Also published as: DE4239531A1; WO1994012518A3

Abstract

The invention concerns a ribooligonucleotide of the formula I or II, in which R1 is a hydroxyl or phosphate group or at least one 3', 5'-nucleotide bisphosphate; R2 is a nucleobase; R3 is the molecule to be coupled to the nucleic acid and R4 is a hydrogen atom or a nucleoside, m being a number between 1 and 100, in particular between 2 and 5; n1 being either 0 or a number between 1 and 30, in particular 1, and n2 being either 0 or a number between 1 and 30, in particular 1. The ribooligonucleotide is suitable for use in coupling marker compounds, in particular fluorescent markers but also proteins, in particular nucleases, to nucleic acids, using RNA ligases as catalysts. This modified ribooligonucleotide can be used for (a) hybridization processes, (b) investigations of the structure of RNA, (c) studies of the interactions between proteins and RNA or (d) particular modifications of nucleic acids.

Description

Reagent for coupling various substances to nucleic acids and process for its production

The present invention relates to a modified ribooligonucleotide, that is to say a reagent for coupling at least one molecule to nucleic acids, in particular ribonucleic acids (RNA), a method for its production, its use and a reagent kit based on this modified ribooligonucleotide. This coupling reagent is a specifically modified ribooligonucleotide, which in principle consists of any number, but at least one ribonucleotide. With the help of this coupling reagent, it is possible to use a variety of labels, in particular fluorescent labels, but also digoxigenin or biotin derivatives, or else other molecules, such as e.g. B. to couple proteins to nucleic acids, especially ribonucleic acids (RNA).

The coupling of a wide variety of molecules to nucleic acids is a widely used method in molecular biology. Such methods are particularly important for labeling nucleic acids. They are used for protein-nucleic acid interaction studies, nucleic acid structure studies, for DNA and RNA sequencing or, for medical diagnostics of enormous importance, in hybridization methods, especially in-situ hybridization methods. Labeling has so far been carried out mainly on a radioactive basis with the aid of isotope-substituted nucleotides and corresponding enzymes. However, since radioactively labeled substances are quite expensive, handling them is not safe and the disposal of radioactive waste is problematic for ecological reasons, efforts have been made for some time to use alternative (i.e. non-radioactive) labeling options.

Probably the most commonly used labeling technique for RNA uses a 3 \ 5 "bisphosphate, normally [5 - ³² P] pCp (cytidine 3 \ 5" bisphosphate), but pCp can also be mashed with isotopes other than ³² P. With catalysis by an RNA ligase, this pCp is coupled to the 3 end of an RNA. The product This reaction is an RNA molecule that is extended by one nucleotide and bears a radioactive label at the 3 "terminus. This method only allows the radioactive labeling of RNA.

An enzymatic method for fluorescent labeling of tRNA and oligodeoxynucleotides is described in the journal "Nucleic Acids Research" 12 (4) 1791-1810 (1984). A modified pdCp is used as coupling reagent, in which an oxygen atom of the 3 "phosphate group is replaced by a sulfur atom, so that a molecule of the form pdCp (S) is formed. A fluorescent molecule, a biman, is bound to this sulfur atom. By means of T4 RNA ligase, this molecule is then bound to the 3 "end of a tRNA, as well as to various synthetic oligodeoxynucleotides that are used as models for deoxynucleic acids. The disadvantage here is that the choice of the fluorescent marker is very limited, since only a few of the customary markers can be bound to the sulfur.

In the "Journal of Histochemistry and Cytochemistry" 29, 227-237 and 238-246 (1981), a method is described with which suitable labels bind to the resulting aldehyde groups by periodate oxidation of the adjacent 2 ", 3" hydroxyl groups of a terminal riboside to let. The side reactions that occur are disadvantageous in this method. A large part of the product is lost due to the inevitable ß-elimination. Satisfactory results are obtained only when using hydrazides, since in this case no ß-elimination occurs: A further disadvantage is that the substances used as donors have to be subjected to a very complex chemical pretreatment. In addition, chemical labeling methods of this type have the major disadvantage that they are less specific than enzymatic methods in general. Due to the high specificity of enzymes, side reactions are largely excluded from the outset.

Progress is already being made by the further labeling method for RNA described by RW Richardson and RI Gumport in the journal "Nucleic Acids Research" 11 (18), 6167-6184 (1983). It is proposed to use β-substituted ADP derivatives of the form Ado-5 "PP-X to catalyze the T4 RNA ligase enzyme to form the PX part of the molecule to form a 3" phosphodiester bond to the 3 "end of one X can be, for example, a fluorescent marker such as fluorescein or rhodamine, and one disadvantage of this method is that the synthesis of the ADP derivatives is relatively complicated and time-consuming the number of marker substances that can be used is also very limited in this method.

A further advance is in the method by Zuckermann et al. (see "Nucleic Acids Research" 15 (13), 5305-5321 (1987)). The authors describe a method for the synthesis of DNA oligomers with a free 3 "thiol group, which can be labeled with suitable samples. The SH group, which is intended for binding the marker, acts as a binding molecule to the column material during the synthesis. However, this means, for example, that only one marker can be bound to the oligonucleotide Another disadvantage is that a free SH group is relatively sensitive, so that longer storage of the unlabeled oligonucleotide is critical.

The object of the present invention is to develop a special ribooligonucleotide, i.e. H. to provide a reagent for coupling at least one molecule, more generally considered different substances, in particular a fluorescent marker, a biotin derivative, a digoxigenin derivative or a protein to nucleic acids, in particular ribonucleic acids (RNA), which avoids the disadvantages mentioned, i.e. its use on enzymatic Implementations is based and therefore has a high specificity that is easy to carry out and that allows many different substances to be coupled to nucleic acids. Furthermore, it is the object of the present invention to provide specific possible uses for the modified ribooligonucleotide, as well as a reagent kit based on this ribooligonucleotide.

This object is achieved in that a coupling reagent with a structure according to formula (I) according to claim 1 or (II) according to claim 2 or (III) according to claim 3 is provided. The molecule R ₃ (or the molecules) which is to be coupled to the nucleic acid is bound to this coupling reagent and by means of a simple method with the one to be labeled

Nucleic acid coupled. The molecule (R ₃ ) is attached to a

Nucleic acid is coupled, is preferably a fluorescent marker, for example a fluorescein derivative, but can also be a digoxigenin or biotin derivative or a protein, for example. Regardless of the nature of this molecule R ₃ , it is referred to below as a marker for the sake of simplicity. In principle, the introduction of a radioactive isotope is also possible, in contrast to the pCp conventionally used for radioactive labeling with the Coupling reagent according to the invention also "exotic" isotopes, such as ¹²⁵ J can be introduced.

The now good availability of fluorescein-labeled RNA as a result of the coupling reagent according to the invention also makes it possible to carry out RNA sequence analyzes automatically in analogy to DNA sequence analyzes with the aid of DNA sequence analysis devices. Automatic DNA sequence analysis is described in detail in Ansorge et al., Magazine "Nucleic Acids Res". 16, 2203 (1988).

The option of specifically coupling proteins to nucleic acids with the help of modified ribooligonucleotides opens up completely new possibilities in the manipulation of nucleic acids. By coupling corresponding nucleases to ribooligonucleotides, which are used as a probe, it is possible to cleave nucleic acids at a precisely defined location. U. Englisch and D.H. Gauss explicitly emphasizes: "There is a need for the artificially site-specific cleavage of ever different RNA or DNA single strands." [Chemically modified oligonucleotides as probes and agents; Journal "Angewandte Chemie" 103 (6), 629-739 (1991)]. The coupling of a DNA-cleaving enzyme to a ribooligonucleotide is particularly advantageous, since this prevents the enzyme from destroying the oligonucleotide to which it is coupled, which acts as a probe.

Further specifications with regard to the RNA according to the invention are the subject of the dependent claims.

In other words than those used in the main claim, the coupling reagent according to the invention consists of a ribooligonucleotide of any length in which a reactive group, in particular an amino group, is bonded directly or via a spacer group to one or more 3 ″ phosphate groups. A sulfhydryl group can also be used as a reactive group "However, it is more sensitive and does not react with as many substituents as an amino group. With the minimal number of nucleotides with which it functions according to the invention, the coupling reagent has, for example, the form according to claim 2:"

where each

Ri is a phosphate group

R _{2 is} a nucleobase, especially cytidine

R _{3 is} the molecule to be coupled and

R "represent a hydrogen atom, and where m = 1, n _x = 1 and n ₂ =

When using the bases adenine, guanine or uracil, a coupling reagent which also functions according to the invention is likewise obtained, but the conversion rates of the coupling reaction are up to 50% lower than when using cytidine. The ribooligonucleotide can be extended on its 3 "phosphate group with further nucleotides, which can also be substituted.

In a further embodiment, the alkyl chain used as spacer contains no phosphate group and therefore also no R ₄ radical. A hydrogen atom is then instead bonded to the carbon atom of the alkyl chain to which a phosphate group is bonded according to claims 1 and 2. An extension of the coupling reagent in the manner described above is then not possible and the coupling reagent has only a single marker molecule R ₃ .

One of the advantages of using a modified ribooligonucleotide as a coupling reagent is that it can be coupled enzymatically to the 3 "end of a nucleic acid using an RNA ligase, especially the T4 RNA ligase. The minimum requirements of this enzyme must be Substrate is a 3 ", 5" ribonucleoside bisphosphate. Ribooligonucleotides which contain such a group at their 5 "end are bound by the RNA ligase to the 3" - OH group of any ribonucleic acid. The reagent can also be coupled to a DNA; all that is required is to attach a ribonucleotide to the 3 "end of the DNA before coupling.

In a preferred embodiment, the reactive group which binds the marker is bonded to the 3 "phosphate group of the coupling reagent via a spacer containing phosphate. The spacer is an alkyl chain with a length of between 1 and 30 carbon atoms, to which a phosphate group can be bound. The coupling reagent can be extended further at this phosphate group of the spacer: Either by attaching another nucleoside to which a marker can in turn be bound (see formula I), or by directly introducing further marker molecules with a spacer (see formula II) .

Since the length of the ribooligonucleotide can be selected as required and there is in principle the possibility of binding markers to any or all of the phosphate groups with the exception of the 5 "terminal phosphate group, a reagent is available with the coupling reagent according to the invention with which nucleic acids can be used in one step can be coupled with several markers, which, for example, gives a stronger signal, particularly in connection with in situ hybridization methods, which is a major advantage of the coupling reagent according to the invention, since it is very complex to obtain a signal amplification in conventional labeling methods.

The coupling reagent is synthesized analogously to the automatic DNA synthesis. It is essential to the invention that the coupling reagent can be synthesized automatically with the aid of conventional DNA synthesizers, the synthesis cycle being suitably modified to the requirements of RNA synthesis.

This means that ribonucleotides are used as building blocks instead of deoxyribonucleotides and uracil is used instead of thymidine. Furthermore, the 2 "-OH group is protected by a protective group, for example a silyl group, during the reaction. Finally, the reaction time in RNA synthesis is approximately 10 minutes compared to approximately 3 minutes in DNA synthesis.

The ribooligonucleotide is synthesized according to the phosphoramidite method, which is described in detail by T. Atkinson and M. Smith (in: Oligonucleotide Synthesis; A Practical approach; MJ Gait (ed.) IRL Press, Oxford, Washington DC, 1984) on the solid phase from 3 "to 5".

A reactive amino group is preferably introduced with the aid of a special amination reagent of the form (l-dimethoxytrityloxy-3-fluorenylmethoxycarbonylamino-propan-2-yl) - (2-cyanoethyl) - (N, N-diisopropyl) - phosphoramidite (for example from the company Glen Research [Sterling, Vancouver, Canada] available as 5 "-branched modifier C3). Using this amination reagent results in a coupling reagent with a 3" -terminal nucleoside. This can be modified further, for example by oxidizing the 2 "- and 3" -OH groups to aldehyde groups. These aldehyde groups can react with amino groups to form a Schiff base. If the aldehyde groups are reacted with corresponding amino groups of enzymes, the enzyme is bound to the oligonucleotide.

If the terminal nucleoside is not modified, there is a risk that the 3 "OH group of this nucleoside reacts with the 5" phosphate group of the same type of molecule. In order to avoid such self-ligation of the ribooligonucleotide according to the invention, whereby a small percentage of the coupling reagent can be inactivated, the nucleoside can be split off at the 3 "end by means of β-elimination and the self-ligation can thereby be prevented.

Alternatively, the reactive amino group can also be introduced with an amination reagent of the form (l-dimethoxytrityloxy-3-fluorenylmethoxycarbonylamino-propane-2-succinoyl) - aminoalkane (available, for example, from Glen Research as a 3 "amino-modifier-CPG), where directly a 3 "phosphate group according to formula (I) or (II) results. In this case, the β-elimination is saved if you do not want a terminal nucleoside.

Various substances (R ₃ ) which easily react with an amino group, such as fluorescent markers, for example a fluorescein derivative, or digoxigenin or biotin derivatives or else proteins, in particular nucleases, are then linked to the introduced amino group. Activated substituents can exist as isothiocyanates, isocyanates, as N-hydroxysuccinimide esters, p-nitrophenyl esters, sulfonic acid chlorides, triazine chlorides or as acid azides. O

The following substances are preferably used as markers: fluorescein succinimide ester, NBD-F (4-fluoro-7-nitrobenz-2-oxa-1,3-diazole), tetramethylrhodamine 5 (6) isothiocyanate, Texas red (sulphorhodamine-101-acid chloride) , Biotin succinimide ester, digoxygenin succinimide ester.

In addition to such marker substances, proteins, in particular nucleases, can also be bound to the coupling reagent. In addition to the method already described above, this "crosslinking" of an enzyme to an oligonucleotide can in principle be carried out in the following ways: on the one hand, a free sulfhydryl group can be incorporated into the enzyme by incorporating a cysteine residue (see R. Zuckermann et al., Journal "J. Am. Chem. Soc. "110, 1614 (1988)), which then reacts with the sulfhydryl group of a so-called hetero cross-linking reagent (eg N-succinimidyl 3- (2-pyridyldithio) propionate) to form a disulfide bridge. The succinimidyl residue of the hetero crosslinking reagent reacts with the amino group of the coupling reagent, causing the enzyme to cross the

Crosslinking reagent is bound to the coupling reagent. Another way of introducing a free SH group into the corresponding enzyme is to chemically convert an amino group into an SH group (see Carlsson et al., Journal "Biochem. J". 173, 723-737 (1978)) . The actual cross-linking takes place as described above. A third possibility of binding an enzyme to the coupling reagent is to use a homo cross-linking reagent which, in contrast to the hetero cross-linking reagent mentioned above, has two succinimidyl residues. It reacts both with the amino group of the coupling reagent and with an amino group of the enzyme.

The introduction of a phosphate group at the 5 "terminus of the ribooligonucleotide, which is necessary for the reaction of the ribooligonucleotide with the nucleic acid to be labeled, is preferably carried out by means of a phosphorylation reagent of the form: 2- [2- (4,4" -dimethoxotrityloxy) ethylsulfonyl ] ethyl- (2-cyanoethyl) - (N, N-diisopropyl) - phosphoramidite (available from Glen Research as a chemical phosphorylation reagent). Such a 5 "phosphorylation is also possible with other methods, for example using ATP and the enzyme polynucleotide kinase. However, this method is extremely cumbersome and lengthy, especially on a larger scale.

Using the corresponding activated nucleosides and the amination and phosphorylation reagents described above, the coupling reagent according to the invention can be used with the aid of conventional DNA synthesizers are automatically synthesized. The synthesis of a coupling reagent of the form 5 "pCpCp fluorescein is explained in more detail below as an exemplary embodiment.

a) Synthesis of the intermediate 5 "pCpCp. Amino) A

With the aid of the DNA synthesizer 394 from Applied Biosystems [Foster City, California, USA], a modified RNA triplet was first produced from the following building blocks (direction: 5 "- 3") using the phosphoramidite method:

5 "1.) Phosphorylation reagent (Chemical Phosphorylation Reagent) 2.) RiboC phosphoramidite 3.) RiboC phosphoramidite

4.) Amination reagent (5 "-branched modifier C3) 3" 5.) RiboA-CPG (control pore glass)

The reagents were obtained from Glen Research, but are also available from other companies.

For the synthesis of approx. 1 μmol pCC (amino) A, 30 mg RiboA-CPG are filled into a column and this is then sealed. The RNA synthesis cycle is started. The setting on the DNA synthesizer is: Tr off, auto. This means that the protective groups are split off and the product is split off from the carrier. For this purpose, the column after the reaction is rinsed with ethanol: NH _3/1: 3.

Incubation of the solution obtained for 12 h at 55 ° C. completes the removal of the protective groups.

The product thus obtained is lyophilized and then taken up again in 0.2 ml of 1 M tetraethylammonium fluoride in tetrahydrofuran. It is then incubated for 6 h at room temperature, as a result of which the protective groups on the ribose are split off. 0.3 ml of 1 M ammonium acetate solution, pH 5.3, is then added to this solution and diluted with 5 ml of water. For the purification of the ribooligonucleotide by means of ion exchange chromatography, this solution is placed on a Sephadex A25 ion exchange column (Pharmacia [Uppsala, Sweden]) equilibrated with 50 mM triethylammonium acetate (TEAA) with a volume of 1 ml. This column is then washed first with 0.2 M TEAA, then with 0.5 M TEAA and the product is then eluted with 2 M TEAA. The product thus obtained is lyophilized, taken up in 70% ethanol and again lyophylized. Finally, the modified ribooligonucleotide in 100 ul water dissolved and its concentration determined by spectrophotometric determination of the absorption at 260 nm (1 OD = 30μg / ml). The substance can be stored at -20 ° C or lyophilized for a long time without loss.

b) Labeling of the ribooligonucleotide with fluorescein

The incubation medium contains:

- 0.1 μmol of the modified ribooligonucleotide in 1 ml of water

0.1 ml of 1 M sodium hydrogen carbonate, pH 8.8-3 mg of fluorescein succinimide ester (FLUOS, Boehringer [Mannheim,

FRG]) in 150 l of dimethylformamide

This mixture is incubated for 18 h at room temperature in the dark. After 18 h, 0.2 ml of 1 M ammonium acetate solution, pH 5.3, are added and this solution is then lyophilized. Free fluorescein is removed from the dried product by extracting four times with 1 ml of ethanol. The product is then taken up in 100 μ \ 2 M TEAA, pH 7 and again lyophilized. The cleaning is then carried out chromatographically, as described under a).

c) Removal of the 3 "terminal adenosine residue by periodate oxidation

In order to protect the 3 'end from self-ligation, the 3'-terminal adenosine residue is removed by periodate oxidation. The incubation medium contains: - 50 μ \ labeled ribooligonucleotide (from b)

- 1 μ \ 3 M sodium acetate, pH 4.8

- 50 μ \ 50 mM sodium periodate

Incubation takes place at 0 ° C for 1 h. Excess periodate is destroyed by adding 50 μ \ ethylene glycol and further incubation for 30 min at 0 ° C. Subsequent addition of 150 μl of 0.5 M lysine (or any primary amine), pH 9.3 and incubation for 4 h at room temperature removes the ribose together with the attached adenine base by means of β-elimination. The reaction solution is now diluted to 1.5 ml with water and, as described under a), applied to a Sephadex A 25 column, washed, eluted, the eluate lyophilized, dissolved in water and its concentration determined. The coupling reagent now has the form according to formula II (claim 2), where R _α 3 ", 5" -cytidyl bisphosphate, R ₂ cytidine, R _{3 is} a fluoresceinyl group and R _{4 is} a hydrogen atom and where n ₂ = 1, n ₂ = 1 and m = 1. In this form, the coupling reagent is used for coupling to nucleic acids.

Coupling of the coupling reagent to nucleic acids

An enzyme, namely an RNA ligase, preferably the T4 RNA ligase, catalyzes the bond between the 5 "phosphate end of the coupling reagent and the 3" OH end of the nucleic acid to be modified. The reaction is carried out in a special RNA ligase buffer which contains magnesium chloride, an organic solvent, ATP, coupling reagent, the nucleic acid to be modified and the RNA ligase at a slightly alkaline pH. This mixture is incubated at temperatures between 0 and 10 ° C. for 4 to 24 h and the modified nucleic acid is then extracted with phenol and precipitated with ethanol.

As an example, the labeling of an RNA with fluorescein is carried out below using the coupling reagent, the synthesis of which was described in the example above.

The total volume of the incubation medium contains 30 μ:

- 15 μl double-concentrated RNA ligase buffer (100 mM Hepes / KOH, pH 7.8; 40 mM magnesium chloride; 7 mM dithiothreitol; 20 μg / ml bovine serum albumin; 20% dimethyl sulfoxide)

- 3 ul 1 mM ATP

- 1 μl 180 μM coupling reagent (to which fluorescein is bound)

- 5 μg RNA (or less)

- 20 U T4 RNA ligase

This mixture is incubated at 4 ° C for 8 h. The labeled RNA is then obtained by phenol extraction and subsequent precipitation with ethanol. Use of the coupling reagent

A main application of the coupling reagent according to the invention is the coupling of labeling substances (R ₃ ), in particular fluorescent markers, to nucleic acids. In practice, the straightforwardness of the method of binding marker substances to the coupling reagent and the coupling reagent to a nucleic acid makes it possible, for example in the form of a kit, to provide the user with the still unmodified coupling reagent and separately various marker substances. The user can then easily produce the coupling reagent as required and in the required modification as required. Since the coupling of the modified coupling reagent to the nucleic acid to be labeled is an enzymatic method, the user has a simple possibility of coupling any nucleic acids with markers of his choice very gently and with high specificity.

Claims

1. Ribooligonucleotide for coupling at least one molecule, for example a fluorescent marker or a biotin derivative or a digoxigenin derivative or a protein, to nucleic acids, in particular ribonucleic acids (RNA), characterized by the following structural formula (I):

where each

R _{x is} a hydroxyl group or phosphate group or at least one

3 ", 5" nucleotide bisphosphate,

R _{2 is} a nucleobase,

R _{3 is} the molecule to be coupled and R. is a hydrogen atom or a nucleoside, and where m is a number between 1 and 100, in particular between 2 and 5, and _{x is} either 0 or a number between 1 and 30, in particular 1, and n, are either 0 or a number between 1 and 30, in particular 1. Ribooligonucleotide for coupling at least one molecule, for example a fluorescent marker or a biotin derivative or a digoxigenin derivative or a protein, to nucleic acids, in particular ribonucleic acids (RNA), characterized by the following structural formula (II):

where each

R _{x is} a hydroxyl group or phosphate group or at least one

3 ", 5" nucleotide bisphosphate, R _{2 is} a nucleobase,

R _{3 is} the molecule to be coupled and

R _{4 represents} a hydrogen atom or a nucleoside, and where m is a number between 1 and 100, in particular between 2 and 5, and n _j either 0 or a number between 1 and 30, in particular 1, and n, either 0 or a number between 1 and 30, in particular 1

3. Ribooligonucleotide according to claim 1 or 2, characterized in that the alkyl chain serving as a spacer contains no phosphate group with R ₄ attached thereto and instead contains a hydrogen atom (formula III).

4. Ribooligonucleotide according to one of claims 1 to 3, characterized in that the nucleobase (R ₂ ) is an adenine, guanine, uracil or

Is cytidine. 5. Ribooligonucleotide according to one of claims 1 to 4, characterized in that the molecule to be coupled R _{3 is} a fluorescent marker, in particular a fluorescein derivative, a biotin derivative, a digoxigenin derivative, or a protein, in particular a nuclease.

6. A process for the synthesis of a ribooligonucleotide according to one of claims 1 to 3, characterized in that the synthesis of a ribooligonucleotide modified with one or more amino groups is carried out with a DNA synthesizer with a modified synthesis cycle, the components:

- phosphoramidites

- amination reagents

- Phosphorylation reagent are used and the desired marker (R ₃ ) is then bound to this modified ribooligonucleotide using methods known per se.

7. Use of riboohgonucleotides according to one of claims 1 to 5 a) for the production of probes for hybridization processes, in particular in situ hybridization processes or b) for RNA structural studies or c) for protein-RNA interaction studies or d) for the production of oligonucleotide enzyme -Complexes that are suitable for the targeted processing of nucleic acids.

8. Reagent kit for coupling molecules to nucleic acids, in particular

Ribonucleic acids, consisting of: a) reagent according to one of claims 1 to 5, wherein R _1? R, and optionally R _{4 have} the meaning given above and R ₃ instead of the one to be coupled

Molecule is a hydrogen atom b) marker substance or nuclease c) RNA ligase, in particular T4 RNA ligase