Methods for Labeling Nucleic Acids

Nucleic acid probes can be labeled with tags or other modifications during synthesis. However, purchasing custom oligonucleotide probes (especially RNA) can be quite expensive depending on the modification and whether costly purification services are required. Additionally, the minimum order quantity for modified oligonucleotides is typically much higher than unmodified versions and may be excessive compared to the amount required for the intended application. Because of this, many researchers may choose in-house methods or labeling kits for probe generation.

Numerous reagents are available for quick and efficient benchtop oligonucleotide labeling, and they are most useful for making small amounts of probe or when many different probes with the same label are required (i.e., for mutational analysis). For small-scale probe generation needs, enzymatic methods are an economical method for labeling probes. In contrast, chemical methods are amenable to larger scale reactions. There are enzymatic and chemical methods for creating probes labeled at either the 5′ or 3′ ends of the oligonucleotide as well as randomly incorporated throughout the sequence. The choice of method needed is determined in part by the degree of labeling required and whether the modification will cause steric hindrance that prevents the desired interactions. Typically, nucleic acids hybridization reactions (i.e., northern blotting) benefit from the high specific activity gained through random incorporation of label into a probe. However, assays requiring protein interactions (i.e., gel shift and pull-down assays) require end-labeling to allow protein binding.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase enzyme expressed in certain populations of lymphoid cells. TdT typically adds numerous deoxynucleotides to the 3′ terminus of a DNA strand, but reaction conditions can be optimized such that only 1–3 incorporation events occur. TdT is template independent and not significantly affected by DNA sequence, but DNA structure is important. TdT has the highest activity towards the 3′ end of single-stranded DNA but can also modify the 3′ overhang of double-stranded DNA with lower efficiency. TdT has poor activity towards double-stranded DNA with blunt ends or 5′ overhangs. Common sources of DNA templates modified with TdT include unlabeled, single-stranded PCR primers and double-stranded restriction endonuclease fragments with 3′ overhangs (“sticky ends”, 5′ recessed ends).

TdT is often used to label DNA probes for RACE (Rapid Amplification of cDNA Ends), TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assays and as a method for adding 3′ overhangs to DNA fragments to facilitate cloning. TdT can also be used to label the 3′ end of DNA probes with radioactive and nonradioactive tags for a variety of detection and affinity applications. For example, TdT addition of biotin-11-UTP to the 3′ end of complementary DNA probes is an effective way of creating probes for use in nonradioactive electrophoretic mobility shift assays (EMSA) and DNA pull-down assays. The following graph shows a comparison of TdT incorporation of several different modified nucleotides.

TdT incorporation of modified nucleotides. 30 units of TdT were incubated with a 48-bp oligonucleotide and an equimolar mix of the modified nucleotide with three other nucleotides for 4 hours at room temperature. The reaction products were analyzed on a 20% TBE pre-cast gel stained with Invitrogen SYBR Gold Nucleic Acid Gel Stain. TdT incorporation of different modified nucleotides is compared.

T4 RNA ligase is an enzyme coded for in the genome of the T4 bacteriophage. T4 RNA ligase catalyzes the attachment of a terminal 5′-phosphate to a terminal 3′-hydroxyl group on RNA. T4 RNA ligase is template independent but requires single-stranded RNA and ATP. Though the primary substrate for T4 RNA ligase is RNA, reaction conditions can be optimized for single-stranded DNA molecules; however, efficiency is very low.

T4 RNA ligase is used for labeling the 3′ end of RNA with [5′ ³²P]pCp (cytidine-3',5'-bis-phosphate), modifying mRNA for cDNA library generation and performing 5′-RACE. T4 RNA ligase can also be used to 3′ end-label RNA with nonradioactive tags using an appropriately modified nucleoside 3',5'-bisphosphate.

Chemical process for labeling the 3′ end of RNA using T4 RNA ligase.

T4 polynucleotide kinase (T4 PNK) is an enzyme coded for in the genome of the T4 bacteriophage. T4 PNK transfers an organic phosphate from the gamma position on ATP to the 5′-hydroxyl group of DNA and RNA. The wild-type enzyme also has 3′-phosphatase activity. T4 PNK ligase is template independent and modifies single-stranded polynucleotides and 5′ overhangs efficiently. Blunt-ended and 5′ recessed ends can be modified with reduced efficiency.

T4 PNK is used primarily for labeling the 5′ ends of polynucleotides with radioactive phosphate from isotope-modified ATP. PNK is more efficient at modifying short overhangs and blunt-end fragments than TdT or T4 RNA ligase. While it is possible to perform phosphate-exchange reactions, PNK labeling is most efficient when the 5′ end of the target molecule has been dephosphorylated. Another common use of PNK is the 5′ phosphorylation of synthetic polynucleotides (i.e., DNA primers) to facilitate cloning. The Invitrogen KinaseMax 5' End-Labeling Kit allows the efficient end-labeling of DNA or RNA to high specific activity with T4 polynucleotide kinase and [gamma-³²P] ATP, or quantitative phosphorylation of 5' ends using unlabeled ATP. The kit includes sufficient reagents for 30 reactions.

5’ end-labeling reactions with T4 PNK. Comparison of 5’ end-labeling reactions using the standard and KinaseMax forward reaction buffer for a 24-mer DNA and 18-mer RNA oligonucleotide. The DNA and RNA oligonucleotides were labeled in 10-ul reactions using 10 units of T4 polynucleotide kinase, 25 pmol [gamma-32P] ATP, and forward reaction buffer. Use of this kit results in greater yield of labeled oligonucleotides.

DNA polymerases are a family of enzymes that create deoxyribonucleic acid polymers by catalyzing the joining of the 5′-phosphorylated end of a deoxyribonucleotide (monomer) to the 3′-hydroxyl end of an existing DNA strand (DNA elongation) or primer (primer extension). DNA polymerases are template dependent but not sequence dependent. To synthesize DNA, the 3′-OH end of an existing DNA strand must be annealed to a complementary strand of DNA. Viral reverse transcriptase enzymes are an exception to this rule and make use of DNA or RNA primers and a RNA template for the synthesis of complementary DNA. The DNA polymerase will synthesize a new DNA strand through elongation of the existing 3′-OH end, adding individual nucleotides complementary to the template strand being read. DNA polymerases are used for a variety of lab purposes from cloning to sequencing. Applications requiring the amplification of a specific fragment of DNA are performed with the aid of heat stable enzymes cloned from thermophilic organisms (e.g., Thermus aquaticus (Taq), Bacillus stearothermophilus (Bst), Thermococcus litoralis (Vent)) using the polymerase chain reaction (PCR). For applications in which amplification is not required, DNA polymerases from mesophilic organisms (e.g., E. coli (Klenow) and bacteriophage (T4, T7)) may be used depending on the length of the DNA to be synthesized and the degree of replication fidelity that is required.

Probes generated with the aid of DNA polymerase are most commonly made by the random incorporation of modified nucleotides during the DNA replication process, which can be done by PCR or simple primer extension reactions. Probes generated in this manner have high specific activity and allow the detection of small quantities of target. Traditionally, this method required radioactive nucleotides to generate probes; however, biotin, fluorophores and other nonradioactive tags can be used if the modification does not interfere with the polymerase elongation reaction (except for terminator sequencing applications). For applications requiring a lower specific activity or when targeted labeling is desired, DNA polymerase can be used to specifically label the ends of a DNA probe. To label the 5′-end of a DNA probe, a 5′ end-modified primer must be used. This method is particularly useful when adding affinity tags or fluorophores to DNA probes that are too long for efficient automated synthesis. This method is ideal for 5′ end-labeling by PCR or simple primer extension reactions.

To label DNA probes at or near the 3′-end, DNA polymerase can be used to incorporate one or more modified nucleotides into the end of a double-stranded probe with a recessed 3′-end. This "fill-in" reaction can be performed with staggered annealed primers, restriction fragments or any other double-stranded DNA molecule in which a complementary DNA sequence can be annealed and used as the template for the extension of a 3′-end. Klenow fragment (E. coli DNA polymerase I) is commonly used for fill-in reactions and can even be used to 3′ end-label RNA molecules when hybridized to a DNA primer producing a 3′ recessed-end.

Dot blot with biotin-labeled DNA using Klenow fragment. The Thermo Scientific Biotin DecaLabel DNA Labeling Kit, which includes Klenow fragment, exo-, was used to biotin-label Lambda DNA/HindIII fragments. The biotin-labeled DNA was then used as a hybridization probe in a dot blot of the homologous DNA on a SensiBlot Plus Nylon Membrane and developed with the Thermo Scientific Biotin Chromogenic Detection Kit. Use of this kit results in high yields of biotin-labeled DNA due to the Klenow fragment, exo-.

RNA polymerases are a family of enzymes that create ribonucleic acid polymers by catalyzing the joining of the 5′-phosphorylated end of a ribonucleotide (monomer) to the 3′-hydroxyl end of a previously incorporated ribonucleotide. RNA polymerases are template dependent and sequence dependent, requiring a promoter sequence within the template DNA in order to initiate binding of the enzyme and, depending on the host system, various cofactors are required for RNA transcription to proceed on the single-stranded template.

RNA polymerases are used for a variety of lab purposes, from the in vitro synthesis of mRNA to the generation of probes for hybridization and binding assays. Probes generated with the aid of RNA polymerase are made by the random incorporation of modified nucleotides during the transcription process. This is typically done by cloning a cDNA or other template sequence into a plasmid containing one or more RNA polymerase promoter sequences. RNA probes generated in this manner have high specific activity and are most commonly made with radioactive nucleotides, although biotin and other tags are also available. Thermo Scientific Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase with strict specificity for its respective double-stranded promoters. It catalyzes the 5'-to-3' synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter.

T7 RNA polymerase promoter sequence. A linear DNA template with this promoter sequence can be used with T7 RNA polymerase to in vitro transcribe labeled RNA probes. The +1 position indicates the first nucleotide that is incorporated into the RNA during transcription. The bases at positions +1 through +3 are critical for transcription and must be G and 2 purine bases, respectively. Use of T7 RNA polymerase with this promoter sequence can result in the generation of labeled RNA probes.

Meta- and ortho-periodates (IO₄ and IO₆, respectively) are anions formed from iodine and oxygen and commonly found as salts with potassium (e.g., KIO₄) or sodium (e.g., NaIO₄). In solution, periodate cleaves the bonds between adjacent carbon atoms having hydroxyl groups (vicinal diols or cis-glycols), creating two aldehydes groups. The resulting aldehyde groups are spontaneously reactive toward primary amine-containing molecules and surfaces. Aldehydes can be used in two types of coupling reactions with either primary amine- or hydrazide-activated tags. Primary amines react with aldehydes to form Schiff bases, which readily hydrolyze and must be stabilized through their reduction to secondary amine bonds with sodium cyanoborohydride (NaBH₄). Hydrazide-modified molecules also spontaneously react with aldehydes but form fairly stable hydrazone linkages, making the reaction much more efficient. The addition of sodium cyanoborohydride will increase the reaction efficiency further and increase bond stability over time and changes in pH.

Because of its mild oxidative properties, sodium meta-periodate is often used as the oxidizer of protein carbohydrates to generate reactive aldehyde groups for either detection or chemical conjugation procedures. The vicinal diols of ribose in RNA nucleotides can also be cleaved with periodate, enabling this method to be used to add a single 3′-end label to RNA but not DNA.

Cleavage of RNA by sodium meta-periodate.

Carbodiimides are functional groups (RN=C=NR) that are typically used in organic synthesis to activate the formation of amide or phosphoramidite linkages between primary amines (RNH₂) and carboxylate (RCOOR′)- or phosphate (R-PO₄)-containing molecules, respectively. Unlike most crosslinkers, carbodiimides do not become part of the final crosslink between the molecules and thus do not add any additional chemical structure to the resulting products. EDC (EDAC, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) is a water-soluble carbodiimide preferred for use in aqueous reactions in the 4.0 to 6.0 pH range, where the reaction byproducts can be easily removed by dialysis or precipitation of the conjugation products.

In reactions with carboxylic acid groups, EDC forms an active O-acylisourea intermediate that is easily displaced by nucleophilic attack from primary amino groups in the reaction mixture. The same reaction can be performed with phosphates (i.e., 5′ phosphate of oligonucleotides), although imidazole must be included in the reaction to obtain efficient conjugation. Because the 5′ phosphate is required, synthetic oligonucleotides must first be treated with a kinase. With that minor exception, EDC-mediated conjugations are an economical means for coupling both RNA and DNA to nearly any other primary amine-containing molecule or surface.

5′ phosphate activation by EDC and imidazole.

Random chemical labeling of nucleic acids can be accomplished by various means. Because these methods label at random sites along the length of a DNA or RNA molecule, they allow a higher degree of labeling to be achieved than end-labeling techniques. However, one disadvantage of these methods is that the nucleotide bases are directly modified, which will reduce or prevent base-pairing between complementary strands during hybridization experiments. Therefore, it may be necessary to balance the degree of labeling with the hybridization efficiency of the probes in certain experiments.

The most popular chemical random-labeling strategies involve the use of photoreactive labeling reagents or the Universal Linkage System (ULS, KREATECH Biotechnology B.V.). Two types of photoreactive label reagents are used for nucleic acids: phenylazide- and psoralen-based. When the phenylazide functional group is exposed to UV light, it forms a labile nitrene that can insert nonspecifically into double bonds and C-H and N-H sites via addition reactions, provided that more reactive nucleophilic (e.g., primary amines) are not present. Molecules containing the psoralen functional group can be used to label double-stranded DNA or RNA. The psoralen ring structure effectively intercalates into the double-stranded portions, and exposure to UV light causes a cyclo-addition product to be formed with the 5,6-double bond in thymine residues. The ULS labeling reagents contain the proprietary temperature-activated platinum-based moiety which reacts with guanine bases in RNA and DNA and the side chains of methionine, cysteine and histidine residues of proteins.

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