Solid‐Phase Synthesis of Branched Oligonucleotides

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.

50-mL oven-or flame-dried round-bottom flask with rubber septum Glass syringe and needle, oven dried 2 × 5 cm silica-coated thin-layer chromatography (TLC) plate with fluorescent indicator (e.g., Kieselgel 60 F 254 aluminum sheets) 254-nm UV light source 500-mL separatory funnel Gravity filtration device and filter paper  11. Dry the organic layer over anhydrous Na 2 SO 4 . Add more Na 2 SO 4 if the salt crystals clump together upon swirling.
When the solution is dry, the nonhydrated crystals will float in solution upon swirling.
12. Gravity filter the resulting solution through filter paper into a 250-mL round-bottom flask. Rinse the Na 2 SO 4 crystals with 10 to 20 mL ethyl acetate.
13. Remove the solvent under reduced pressure (i.e., in a rotary evaporator with a water aspirator) to yield the crude product as a yellow oil.
Isolate and characterize product 14. Prepare a 5 × 25-cm glass chromatography column by adding a slurry of 40 g silica gel in 50:47:3 CH 2 Cl 2 /hexanes/triethylamine. Precondition with the same solvent.
15. Dissolve the crude material in a minimum amount of 50:47:3 CH 2 Cl 2 /hexanes/triethylamine and load on column. Perform chromatography at a rate of ∼1 in. solvent/min using a small amount of air pressure (APPENDIX 3E). Collect product in 10-ml fractions in small test tubes.
16. Combine product-containing fractions into a 500-mL round-bottom flask and concentrate to an oil on a rotary evaporator.
17. Remove residual triethylamine by co-evaporating the oil first with 50 mL of 95% ethanol followed by 50 mL diethyl ether, to provide the pure product as a pale yellow foam.
18. Store bisphosphoramidite at -20°C under an inert atmosphere protected from light.
Phosphoramidites are particularly sensitive to UV light; therefore, it is best to store the bisphosphoramidite in a dark bottle (or a bottle covered with aluminum foil) in a -20°C freezer. Under these conditions, the phosphoramidite may be stored for an indefinite period of time. Prior to use, its purity may be verified via TLC analysis. If partial decomposition has occurred, or the coupling reactions with S.3 are poor, the compound should be subjected to chromatography again as described above.
Branched nucleic acid synthesis works very well on commercially available solid supports (i.e., LCAA-CPG) containing 20 to 40 µmol nucleoside per gram support; however, even better yields are attainable on CPG supports with higher loadings (e.g., 90 µmol/g; Fig.  4.14.5). Such supports can be prepared using HATU/DMAP as the coupling reagents (see Support Protocol 1), and are ideal for the synthesis of short-length bNAs (e.g., trimers) since, in this case, high loadings ensure proper distance between the neighboring CPGbound nucleosides (Damha and Zabarylo, 1989).
Prepare columns for synthesis 1. Transfer an accurately weighed amount of 5′-O-(4,4′-dimethoxytrityl)-N-protected-2′-deoxyribonucleoside-or -ribonucleoside-derivatized succinyl-LCAA-CPG for a 1-µmol synthesis to an assembled synthesis column.   Figure 4.14.4 Schematic representation demonstrating the convergent synthesis of V-and Yshaped bDNA or bRNA oligonucleotides on a solid support. The method can also be used for the synthesis of short branched sequences-e.g., tetranucleoside triphosphates, NpA(2′pN)3′pNparticularly when the nucleoside loading on the solid support approaches 90 µmol/g. Abbreviations: A, bisphosphoramidite; Bz, benzoyl; DMTr, 4,4′-dimethoxytrityl; N, any nucleotide (RNA or DNA); TREAT-HF, triethylammonium trihydrofluoride; S.7, linear oligonucleotide; S.8 and S.10, full-length bNAs (V-and Y-shaped); S.9a-b and S.11a-b, unbranched linear failure sequences. P* indicates the presence of a 2′-or 3′-linked phosphate to the adenosine branch point, which is formed by the hydrolysis and oxidation of the residual phosphoramidite during solid-phase synthesis.  Figure 4.14.5 Effect of CPG loading on yield of the Y-shaped bDNA, T 10 A 2′,5′-T10 3′,5′-T10 synthesized via the convergent strategy (see Basic Protocol 2). (A) PAGE analysis of the amount of Y-shaped product (i; S.10) and failure sequences (ii; S.11a-b) formed as a function of CPG loading on a 20% denaturing polyacrylamide gel. P* indicates the presence of a 2′or 3′-linked phosphate. (B) Chart demonstrating the increase in the amount of S.10 (i) with increasing nucleoside-CPG loading. The chart also demonstrates the inverse relationship between the amount of extended isomeric linear failures (ii; S.11a-b) and nucleoside-CPG loading. The percentage oligonucleotide was determined by integration of the HPLC peak areas of the compounds in question (see Support Protocol 3) using gradient 3 (see Table 4.14.4). Repeat steps 5 and 6 Column washing steps: 8 Argon reverse flush 5 9 Acetonitrile to column 30 10 Repeat steps 8 and 9 11 Argon reverse flush 5 12 Argon block flush 5 a Alternatively, CPG may be capped manually using acetic anhydride (see Support Protocol 1).

Supplement 9
Current Protocols in Nucleic Acid Chemistry 4.14.8 Solid-Phase Synthesis of Branched Oligonucleotides 2. Acetylate any underivatized amino and hydroxyl groups on the solid support using an ABI 381A automated DNA synthesizer and the capping cycle given in Table 4.14.1 (APPENDIX 3C).
This step also removes traces of water from the solid support.
Alternatively, see Support Protocol 1 for the manual capping procedure on nucleosideloaded CPG.

Synthesize branched oligonucleotides
3. Weigh out the appropriate amount of DNA (S.4a-d) and/or RNA (S.5a-d) 3′-phosphoramidites ( Fig. 4.14.3) and dilute to the appropriate concentration with anhydrous acetonitrile as indicated in Table 4 5. Place all synthesizer reagents (i.e., activator, capping, oxidant, and detritylation solutions, and acetonitrile) and diluted phosphoramidites (step 3) on the appropriate ports of the synthesizer.
6. Place the BIS-A phosphoramidite bottle on the spare phosphoramidite port (the "X" port on the 381A synthesizer).  4.14.9 Synthesis of Modified Oligonucleotides and Conjugates 8. Perform synthesis in the trityl-off mode according to the synthesis cycle outlined in Table 4.14.3 and utilizing the coupling times recommended in Table 4.14.2. Collect dimethoxytrityl solutions in 15-mL test tubes using an external fraction collector.
Turning the trityl mode off ensures that the last nucleotide at the 5′ end has a free hydroxyl group, which is desirable for purification using anion-exchange HPLC (see Support Protocol 3).  (Table 4.14.4) and (B) 20% denaturing PAGE (see Support Protocol 4). (C-E) Analysis of a successful (C-D) and unsuccessful (E) synthesis of the mixed base Y-RNA 5′-CCCUACUAA 2′,5′-GUAUGCCC 3′,5′-GUAUGCCC by (C) anion-exchange HPLC (see A for conditions) and (D-E) 20% denaturing PAGE. The regioisomeric extended failure sequences (ii) are resolved into two peaks by HPLC (A and C), but appear as one band by gel analysis (B, D, and E). In panel E, the major product is the unbranched 8-mer 5′-GUAUGCCC-3′, which accumulates due to the unsuccessful branching of the bisphosphoramidite. P* indicates the presence of a 2′-or 3′-linked phosphate.
Current Protocols in Nucleic Acid Chemistry Supplement 9 4.14.11 Synthesis of Modified Oligonucleotides and Conjugates 9. Upon completion of the synthesis, dry the CPG by manually conducting an argon reverse flush operation on the synthesizer for 10 min. Alternatively, dry the CPG under a stream of nitrogen or argon, or in a vacuum desiccator for 30 min.
11. Purify the bNAs from failure sequences by anion-exchange HPLC (see Support Protocol 3) or denaturing PAGE (see Support Protocol 4).

Measure branching efficiency by trityl color analysis
12. Dilute the dimethoxytrityl solutions collected after each successive coupling (step 8) with 10 mL detritylation solution using a 50-mL buret.
14. Measure the absorbance of the solution on a UV-Vis spectrophotometer between 450 and 550 nm, and record the absorbance peak at ∼505 nm for the dimethoxytrityl cation. 15. Determine the efficiency of each coupling step using the equation where A x is the absorbance of the trityl cation released at any given step and A x−1 is the release in the previous step.

REGIOSPECIFIC SYNTHESIS OF BRANCHED NUCLEIC ACIDS
The protocol described below outlines the regiospecific and divergent synthesis of bNAs via phosphoramidite chemistry according to the method of Braich and Damha (1997;Fig. 4.14.8). This protocol allows for the synthesis of bNA molecules with different DNA sequences surrounding the branchpoint nucleotide. The methodology requires the use of standard DNA and RNA 3′-phosphoramidites (S.4a-d and S.5a-d) for the synthesis of a linear DNA strand incorporating a single ribonucleotide unit. Once this sequence is assembled, all of the 2-cyanoethyl phosphate-protecting groups are selectively removed by treatment with triethylamine. This step is necessary as phosphotriesters are susceptible to cleavage/modification by the ensuing fluoride treatment. The CPG-bound oligomer is then treated with fluoride ions to cleave the tert-butyldimethylsilyl group at the 2′ position of the ribose unit, from which another chain (2′-branch) can be synthesized. This is accomplished using "inverted" DNA phosphoramidites (deoxyribonucleoside 5′-phosphoramidites; S.6a-d), allowing branch synthesis to occur in the opposite (5′-to-3′) direction. With the exception of the decyanoethylation and desilylation steps, the entire process is conducted using an ABI 381A DNA synthesizer.   Figure 4.14.8 Schematic representation for the divergent and regiospecific synthesis of branched DNA. The branching synthon is a standard RNA phosphoramidite (S.5a-d; Fig. 4.14.3). Abbreviations: 3′-pD, DNA 3′-phosphoramidites (S.4a-d); 5′-pD′, inverted DNA 5′-phosphoramidites (S.6ad); 5′-pD′′, higher concentration (0.3M) of inverted DNA 5′−phosphoramidite as first nucleotide coupled to the ribose branch point; 3′-pR, RNA 3′-phosphoramidite (S.5a-d); DMTr, 4,4′-dimethoxytrityl; TBDMS, tert-butyldimethysilyl.

Supplement 9
Current Protocols in Nucleic Acid Chemistry 4.14.14

Solid-Phase Synthesis of Branched Oligonucleotides
Protocol 3) or denaturing PAGE (see Support Protocol 4), and measuring coupling efficiency by trityl color analysis (see Basic Protocol 2) CAUTION: All solutions required for bNA solid-phase synthesis should be prepared in a well-ventilated fume hood.
For synthesis on the ABI 381A DNA synthesizer, the volume of each phosphoramidite addition to the column is 170 ìL.
The RNA 3′-phosphoramidite is the branching synthon. Any of the four standard RNA 3′-phosphoramidites (A, G, C, or U) may be used depending on the specific branch point to be introduced.
5. Enter the base sequence of the linear oligonucleotide to be synthesized in the 5′-to-3′ direction, where the last entry (3′ nucleotide) corresponds to the nucleoside bound to the CPG.
For example, to synthesize the hypothetical linear oligonucleotide (S.12) shown in Figure  4.14.8, enter the sequence 5′-NNNNNNXNNNNNN-3′, where N is any deoxyribonucleoside phosphoramidite and X represents the branch point of the RNA.
6. Perform synthesis in the trityl off mode according to the synthesis cycle outlined in Table 4.14.3 and utilizing the coupling times shown in Table 4.14.2. Collect the dimethoxytrityl solutions in 15-mL test tubes in an external fraction collector.
Turning the trityl mode off ensures that the last nucleotide at the 5′ end has a free hydroxyl group, which is desirable for purification using anion-exchange HPLC (see Support Protocol 3).
7. Acetylate the hydroxyl group at the 5′ terminus by running the automated capping cycle (Table 4.14.1).
Capping the free 5′-OH is necessary as it ensures that extension from this functional group will not occur during the synthesis of the "'orthogonal" 2′-branch.
Cleave 2-cyanoethyl protecting group 8. Dry the CPG by manually conducting an argon reverse flush operation on the synthesizer for 10 min.
9. Remove the synthesis column from the synthesizer and connect it to a 10-mL disposable syringe filled with 4:6 (v/v) triethylamine/acetonitrile. Slowly push the deprotection solution through the column over a 90-min period.
Deprotection of the 2-cyanoethyl phosphate-protecting group converts the phosphotriester to the more stable phosphodiester, which withstands the conditions required for desilylation in the ensuing step. To ensure complete decyanoethylation, push the solution slowly through the column and then pull in on the syringe slightly in order to displace the CPG beads from the base of the column.
10. Wash the CPG beads extensively with 30 mL acetonitrile followed by 30 mL THF using a 25-ml glass syringe attached to the column via the syringe adapter.

Cleave 2′-O-TBDMS group
11. Push 1 mL of 1 M TBAF in THF through the column over a period of 10 min using a 1-mL disposable syringe.
It is essential to use fresh TBAF. In order to ensure complete desilylation, push the solution slowly through the column and then pull in on the syringe slightly in order to displace the CPG beads from the base of the column. Prolonged treatment with TBAF results in cleavage of the oligonucleotide chain from the solid support (Braich and Damha, 1997).
Incomplete desilylation results in the accumulation of silylated linear DNA (S.12; Fig.  4.15.8), which does not allow branch extension from the 2′ position of the branch point (see Fig. 4.14.9B).
13. Reinstall the column on the synthesizer.

Synthesize 2′,5′-linked branch (S.13)
14. Modify the DNA synthesis cycle such that synthesis step 15 becomes the first step in the cycle.
Steps 1 to 14 (TCA treatment) may be disregarded since the assembled chain lacks a dimethoxytrityl group.
15. Weigh out the appropriate amounts of inverted DNA 5′-phosphoramidites (S.6a-d; Fig. 4 16. Install all the inverted phosphoramidites on the synthesizer and place the first inverted phosphoramidite (0.3 M concentration) on the spare port (the "X" port on the 381A).
17. Enter the linear sequence to be synthesized in the 5′-to-3′ direction, where the last entry corresponds to the first phosphoramidite to be coupled to the 2′-hydroxyl of the branch point.
For example, to synthesize the hypothetical branched DNA oligonucleotide (S.13) shown in Figure 4.14.8, enter the sequence 5′-NNNNNN-3′, where N is the first DNA 5′-phosphoramidite to be coupled to the 2′-hydroxyl group of ribose.
18. Synthesize the 2′-branch in the trityl off mode using the modified synthesis cycle starting from step 15 of Table 4.14.3, and utilizing the coupling times shown in Table  4.14.2, except for the first phophoramidite (0.3 M), which should have a coupling time of 30 min.
19. Upon completion of the synthesis, dry the CPG by reverse flushing the column with argon for 10 min. Alternatively, dry the CPG under a stream of nitrogen or argon, or in a vacuum desiccator for 30 min.
20. Cleave the oligonucleotides from the support and deprotect the amino-and phosphate-protecting groups (see Support Protocol 2).
Supplement 9 Current Protocols in Nucleic Acid Chemistry 4.14.16 Solid-Phase Synthesis of Branched Oligonucleotides 21. Purify the bNAs from failure sequences by anion-exchange HPLC (see Support Protocol 3) or denaturing PAGE (see Support Protocol 4) and measure coupling efficiency by trityl color assay (see Basic Protocol 2, steps 12 to 15). Figure 4.14.9.

PREPARATION OF LCAA-CPG SUPPORTS WITH HIGH NUCLEOSIDE LOADINGS
The method described allows for the rapid derivatization of LCAA-CPG having nucleoside loadings up to ∼90 µmol/g, which is 3 to 4 times the loading found in commercially available solid supports. While commercial samples provide more than adequate yields of bNAs (Damha et al., 1992), those with higher loadings (50 to 90 µmol/g) provide the best results (Fig. 4.14.5). For example, the synthesis of small bNAs (i.e., trimers and tetramers) requires that the CPG be densely loaded so that efficient branching may occur. The protocol below, adapted from the work of Pon et al. (1999) and Damha et al. (1990), allows for the rapid esterification of 5′-O-protected ribonucleosides and deoxyribonucleosides to succinyl-LCAA-CPG. The key condensing reagent is a mixture of HATU and 4-DMAP. 4.14.17
2. Cap the bottle with a septum and add 1 mL anhydrous acetonitrile via a 1-mL syringe and needle.
3. Shake 2 to 4 hr on a wrist-action shaker at room temperature.

Do not stir the slurry with a magnetic stir bar as this will break up the glass beads into fine particles that may clog the frit on the DNA synthesizer.
4. Vacuum filter succinyl-LCAA-CPG into a side-arm filter flask through a sintered glass funnel or Buchner funnel with filter paper.
It has been reported that the free carboxylic groups are inconsequential and do not react during phosphoramidite synthesis (Lyttle et al., 1997). 6. Transfer the CPG to a glass vial and dry under vacuum using in a desiccator attached to a vacuum pump.
The derivatized CPG may be stored indefinitely at room temperature, preferably in a vacuum desiccator.
7. Determine nucleoside loading through the quantitation of the released trityl groups from the support-bound nucleoside (UNIT 3.2).
8. Acetylate the solid support with cap A and B capping reagents on a DNA synthesizer using the capping cycle outlined in Table 4.14.1. Alternatively, perform capping as described in UNIT 3.2.
Supplement 9 Current Protocols in Nucleic Acid Chemistry 4.14.18

COMPLETE DEPROTECTION OF BRANCHED OLIGONUCLEOTIDES (DNA AND RNA)
This protocol describes the steps necessary for cleaving the bNA from the solid support and removing the protecting groups from the heterocyclic bases and sugar-phosphate backbone. The first step is treatment of the solid support with concentrated aqueous ammonia to concomitantly cleave both the bNA from the support and the N-acyl and 2-cyanoethyl phosphate-protecting groups. A subsequent deprotection step with triethylammonium trihydrofluoride (TREAT-HF) cleaves the 2′-O-tert-butyldimethylsilyl (TBDMS) protecting groups from branched oligoribonucleotides (Gasparutto et al., 1992). The desilylated material is then precipitated directly using 1-butanol (Sproat et al., 1995). A procedure for the quantitation of oligonucleotides is also described.
2. Add 750 µL cold 29% ammonium hydroxide and 250 µL of 100% ethanol, screw the cap on tightly, and incubate 24 to 48 hr at room temperature on a wrist-action shaker.
If the branched oligonucleotide sequence contains isobutyryl-protected guanosine nucleotides, then deprotection must proceed for ≥48 hr, room temperature.
The ammonium hydroxide should be relatively fresh (<1 month old). Seal the cap on the ammonium hydroxide solution tightly and store at 4°C.
3. Microcentrifuge 1 min at maximum speed, room temperature, and cool 30 min on dry ice or 1 to 2 hr at −20°C.
The contents must be cooled before the screw cap is released to prevent volatile ammonium hydroxide from boiling over, which could result in loss of product.
7. Pool in a total of 1 mL water. For branched RNAs, use DEPC-treated water that has been autoclaved to remove residual DEPC.

The product is a crude deprotected branched DNA or partially deprotected branched RNA. For RNA, DEPC-treated water should be used in all subsequent steps.
Quantitate oligonucleotide 8. Dilute crude bNA 10 or 100 fold with autoclaved water and measure the absorbance at 260 nm (A 260 ) on a calibrated, double-beam, UV spectrophotometer using autoclaved water as a reference.
If using a single-beam spectrophotometer, a baseline should be run with a sample containing autoclaved water (blank).
9. Determine the concentration of the stock solution using the dilution factor and Beer's Law (A 260 = εbc), where ε is the molar extinction coefficient, b is the UV cell pathlength (typically 1 cm), and c is the concentration of oligonucleotide present.
11. Cover the microcentrifuge tubes with aluminum foil and incubate 24 to 48 hr at room temperature on a wrist-action shaker. Wincott et al. (1995).
A white precipitate should be clearly visible after addition of 1-butanol.
13a. Microcentrifuge 10 min at maximum speed, 4°C, carefully remove the supernatant, and wash the white pellet twice with 500 µL cold 70% ethanol each.

Disruption of the pellet during washing steps could result in loss of sample.
14a. Dry the pellet in a Speedvac evaporator, resuspend in 1 ml autoclaved water, and requantitate the amount of crude oligonucleotide (steps 8 and 9).
Once the 2′-O-TBDMS group has been removed, the fully deprotected RNA is sensitive to nucleases and base hydrolysis, which will cause strand cleavage. In order to prevent this, special considerations for working with RNA (APPENDIX 2A) must be observed. Water used for RNA dissolution and buffer preparation should be of the highest quality available (Milli-Q) and should be treated with DEPC.

For small bNAs (<10-mers):
12b. Quench the desilylation reaction with an equal volume of autoclaved water and evaporate in a Speedvac evaporator. Resuspend in 1 mL autoclaved water and requantitate the amount of crude oligonculeotide (steps 8 and 9).

ANALYSIS AND PURIFICATION OF BRANCHED OLIGONUCLEOTIDES USING ANION-EXCHANGE HPLC
Branched oligonucleotides may be easily and efficiently analyzed and purified from the crude mixture by anion-exchange HPLC as described below (Fig. 4.14.6). bNAs of high purity are attainable (>95%; Fig. 4.14.10). The bNA of interest can be readily isolated from the reaction mixture failure sequences. The resultant product is precipitated directly by the addition of 1-propanol (Sproat et al., 1995). This direct precipitation method works very well for the direct isolation of large bNAs (>10 nt). Smaller bNAs do not precipitate out efficiently and must be further purified by size-exclusion chromatography (or reversed-phase Sep-Pak cartridges) subsequent to HPLC separation. As an alternative to HPLC purification, the bNAs may be purified by denaturing PAGE (see Support Protocol 4).
Characterization of the bNAs is conveniently done via MALDI-TOF-MS as described in UNIT 10.1. The matrix and co-matrix typically used are 6-aza-2-thiothymine (ATT) and dibasic ammonium citrate, respectively (Lecchi et al., 1995).
The branched nature of the molecules may also be confirmed via the yeast debranching enzyme (yDBR), a phosphodiesterase specific to hydrolysis of the 2′,5′-phosphodiester bond of oligonucleotides that contain vicinal 2′,5′-and 3′,5′-phosphodiester linkages (Nam et al., 1994;Ooi et al., 2001). Nucleoside composition analysis of bNA is carried out using snake venom phosphodiesterase (SVPD) according to the method of Eadie et al. (1987;UNIT 10.6). This enzyme cleaves bDNA or bRNA from the 3′ termini yielding 5′-monophosphates, which can then be converted to their constituent nucleosides by in situ treatment with alkaline phosphatase (AP). The resulting nucleoside mixture is analyzed by reversed-phase HPLC as described in UNIT 10.5. Alternatively, bNA can be digested with nuclease P1 from Penicillium citrinum, an endonuclease that cleaves bNA to produce the constituent nucleoside 5′-monophosphates and its branch core trinucleoside diphosphate-i.e., A(2′p5′N)3′p5′N (Damha et al., 1992). The released branched trinucleoside diphosphate structure can be readily synthesized (see Basic Protocol 2) and used as a standard during HPLC analysis of the enzyme digest (UNIT 10.6 (Table  4.14.4). The chromatogram was obtained using the same conditions. 5. Load the sample into the sample loop using an appropriate syringe and inject. Elute bNA at 50°C with a flow rate of 1 mL/min and the gradient and run time specified in Table 4.14.4. Record the chromatogram.
The branched DNA or RNA product should be very well resolved from the extended linear failure sequences and other failure sequences present in the crude mixture. Full-length bNAs elute last (i.e., highest retention time). Typical chromatograms for a 31-mer bDNA and 25-mer bRNA are demonstrated in Figure 4.14.6A and C. Note the excellent separation between the bNA of interest and the failure sequences.
Purify bNAs by anion-exchange HPLC 6. Dissolve 40 to 60 A 260 units crude bNA mixture in 1 mL autoclaved water. Heat and microcentrifuge as in steps 2 to 3.
Loading >60 A 260 units may overload the column and compromise the separation of the bNA from the linear failure sequences.
7. Run the sample as described (steps 4 and 5), but set the detector to 290 nm and collect 1-ml fractions from the peaks of interest in sterile 1.5-mL microcentrifuge tubes.
The detector wavelength is set to 290 nm in order to avoid saturation of the detector signal. If the HPLC is equipped with a detector capable of monitoring dual wavelengths, monitor both the 260-and 290-nm profiles.
The anticipated retention time should be very similar to that obtained during routine HPLC analysis.
8. Pool peak fractions and dry them in a Speedvac evaporator. Add 250 µL autoclaved water.
Lithium perchlorate (LiClO 4 ) is much more soluble in organic solvents than other perchlorate salts, making precipitation easy and efficient, and thus preventing a final desalting step. The DNA or RNA isolated is in its lithium salt form.
10a. Microcentrifuge 10 min at maximum speed, room temperature, carefully remove the supernatant, and wash the white pellet twice with 500 µL cold 1-propanol.
Disrupting the pellet during washing steps can result in loss of sample.
11a. Dry in a Speedvac evaporator, resuspend in 1 mL autoclaved water, and proceed to step 12.
Small bNAs do not precipitate out of solution efficiently.
The alkaline phosphatase may be contaminated with adenosine deaminase, which converts adenosine into inosine. The retention time of an appropriate inosine nucleoside control should be obtained prior to HPLC analysis of the digestion mixture (UNIT 10.6).
15. Dry the sample in a Speedvac evaporator and dissolve residue in 15 µL autoclaved water.
16. Analyze the mixture by reversed-phase HPLC on a C18 column using the mobile phases 0.1 M TEAA, pH 7.0, and acetonitrile with the gradient described in UNIT 10.6.
17. Calculate the relative ratios of nucleoside to branched trinucleotide diphosphate by dividing the area of each peak by the extinction coefficients specified in UNIT 10.6.
The extinction coefficient for the branched trinucleotide diphosphate may be calculated using the oligonucleotide extinction coefficient calculator at http://paris.chem.yale.edu/ extinct.html. The extinction coefficients for bDNA and bRNA are assumed to be the same as their linear counterparts.

ANALYSIS AND PURIFICATION OF BRANCHED OLIGONUCLEOTIDES BY DENATURING PAGE
A method for the analysis and purification of bNAs by denaturing polyacrylamide gel electrophoresis (PAGE) is described (also see UNIT 10.4 and APPENDIX 3B). PAGE is a very convenient way to assess efficiency of bNA synthesis since the molecular weight of oligonucleotides bound on the solid support more than doubles after a convergent branching reaction-e.g., reaction of neighboring decathymidylic acid chains with bisphosphoramidite synthon S.3 to give a 21-unit-long bNA molecule (Fig. 4.14.4 and product ii in Fig. 4.14.6B). This generates a gap between the desired bNA product and its precursor molecules, greatly facilitating the separation process. Any type of standard laboratory electrophoresis equipment may be utilized. Most bNAs of >10 nucleotides in length are very well resolved on a 20% denaturing polyacrylamide gel. If shorter sequences must be purified, better resolution is achieved with a 24% denaturing polyacrylamide gel. Setup and polymerization of the gel along with electrophoretic separation conditions are described. The resultant bands may be visualized by UV shadowing and photographed. A technique for the rapid extraction of oligonucleotides from the gel matrix is also described (Chen and Ruffner, 1996).
4. Place gel in electrophoresis apparatus, removing the comb and bottom spacer, rinse the wells, and prerun the gel 30 min at 500 V, room temperature.
5. Dissolve 0.6 A 260 units deprotected bNA sample in 10 µL formamide loading buffer. If the dissolution process is not immediate, place the sample in a sonicator bath 1 to 2 min or heat the samples briefly at ∼50°C.
6. Load the samples into the wells. Load an equal amount of running dye in the first and last well as an external reference marker. Run the gel at 500 V until the bromphenol blue dye is 3 ⁄ 4 of the way down the gel.
7. Disassemble the glass plates and wrap the gel in plastic wrap. Place the wrapped gel over a 20 × 20-cm silica-coated TLC plate with fluorescent indicator and visualize the bands by shining a handheld UV lamp over the gel. Take a picture of the gel using a camera equipped with a UV filter.
CAUTION: Wear safety glasses to avoid eye burn.
A typical crude bNA reaction mixture will consist of at least three bands as shown in Figures  4.14.6 and 4.14.7. The fastest-migrating band is the linear precursor sequence (S.7;Fig. 4.14.4), followed by the extended isomeric failure sequences (S.9a-b for the synthesis of V-shaped bNAs and S.11a-b for the synthesis of Y-shaped bNAs), and finally the products (S.8 and S.10). If the coupling efficiencies between successive nucleotides is less than optimal, a ladder of failure sequences will be evident below the extended branch failure and linear sequences.
Note that the extended isomeric failure sequences are a mixture of regioisomers (S.9a-b and S.11a-b) that are sometimes resolved into two close-moving bands (this is also evident by HPLC analyses). The slowest of the predominant bands is the bNA of interest. If linear markers are run alongside the purified bNA, one observes that bNA has retarded mobility relative to a linear oligonucleotide of identical composition (length and sequence composition). This is due to the increased frictional effects of the bNA relative to the linear sequences as they move through the highly cross-linked gel environment.
Purify bNAs by denaturing PAGE 8. Assemble and run a preparative gel as for the analytical gel (steps 1 to 6) with the following modifications: a. Increase gel thickness (spacers and comb) to 1.5 cm and scale up volume of gel to 50 mL gel solution, 350 µL APS, and 35 µL TEMED. b. Use up to 100 A 260 units crude bNA dissolved in 100 µL formamide loading buffer. c. Use a comb with one large well to purify 60 to 100 A 260 units, one well of a two-well comb for 30 to 60 units, and one well of a three-well comb for <30 units. 9. Disassemble the glass plates, remove the gel, and wrap it in plastic wrap. Place the wrapped gel over a silica-coated TLC plate and visualize the bands by shining UV light over the gel. If desired, photograph the gel using a camera equipped with a UV filter.
CAUTION: Wear safety glasses to avoid eye burn.
10. Excise the slowest moving band and place the gel piece into a sterile 15-mL tube. Crush the gel piece using a sterile spatula and soak in 3 mL gel extraction buffer.
11. Heat 5 min in a 90°C water bath or heating block and rapidly freeze 5 min in dry ice. Thaw contents rapidly at 90°C and centrifuge 10 min at maximum speed × g in a tabletop centrifuge to settle the crushed gel pieces. Alternatively, after extracting in 3 mL gel extraction buffer (step 10) or water, shake the slurry 12 to 16 hr at room temperature on a wrist-action shaker.
The rapid "crush and soak" method described has been outlined in a paper by Chen and Ruffner (1996).

4.14.28
Solid-Phase Synthesis of Branched Oligonucleotides (1986) by solution-phase phosphoramidite chemistry. Chattopadhyaya and co-workers have focused on several strategies for the synthesis of small bRNA and RNA lariats with self-cleavage properties (Rousse et al., 1994).
The authors' group has been describing in detail a methodology for the solid-phase synthesis of Y-shaped RNAs and DNAs, in which the branch point adenosine is linked to identical oligonucleotide chains via vicinal 2′,5′-and 3′,5′-phosphodiester linkages. Hyperbranched or "dendritic" structures have also been synthesized from this laboratory (Hudson and Damha, 1993;Hudson et al., 1998). The synthetic convergent strategy is based on the discovery that nucleoside 2′,3′-O-bisphosphoramidite synthons react with adjacent solid-support-bound chains yielding symmetric V-like molecules. Synthesis is then continued in the 3′-to-5′ direction from the apex of the V to yield Y-shaped structures. This method was adapted to the synthesis of bRNAs with 2′ and 3′ chains of different base composition (Ganeshan et al., 1995). All other synthetic strategies reported so far for the regiospecific assembly of branched oligonucleotides mimicking the natural lariat structures are based upon solution-phase phosphotriester methods, except for the recent work by Sproat et al. (1994), who use a divergent solid-phase phosphoramidite strategy. This strategy has permitted the synthesis of mediumsized branched oligoribonucleotides; however, it requires the use of nonstandard monomers and branch-point synthons. Given this limitation, and those of the authors' current methods, alternative strategies for the regiospecific synthesis of branched oligonucleotides were sought. This led ultimately to the synthesis of multi-copy single-stranded DNA, a peculiar nucleic acid that is produced in E. coli and M. xantus (Damha and Braich, 1998).
In this unit, general procedures for both convergent and divergent solid-phase synthesis of branched oligonucleotides are presented. The conditions of the branching reactions are crucial to the ultimate success of the overall procedure, and the protocols described in this unit should be followed carefully. In spite of these areas of caution, bNA sequences can now be prepared routinely using, for the most part, commercially available reagents and standard laboratory equipment. The novice should have no hesitation in setting forth!

Compound Characterization
Analysis of crude and purified bNAs is conveniently carried out via anion-exchange HPLC (see Support Protocol 3). The buffer concentration required for the elution of bNA is dependent upon the molecular weight and charge of the molecule being purified (Table  4.14.4). Separation of bNA from failure sequences is greatly facilitated by the relatively large molecular weight of the bNA species. Gel electrophoresis is another convenient way to isolate/purify bNA fragments (see Support Protocol 4). In this case, the bNA of interest exhibits significant retarded mobility relative to the failure sequences as a result of frictional effects as the bNA molecules migrate through the cross-linked gel environment. Further characterization is conducted by MALDI-TOF-MS (UNIT 10.1) using 6-aza-2-thiothymine (ATT) and dibasic ammonium citrate as matrices (Lecchi et al., 1995). Nucleoside composition analysis is conducted via enzymatic hydrolysis with the exonuclease snake venom phosphodiesterase (Eadie et al., 1987;UNIT 10.6) and the endonuclease nuclease P1 (see Support Protocol 3). The yeast debranching enzyme (yDBR), a phosphodiesterase specific to the 2′,5′-phosphodiester bond of oligonucleotides containing vicinal 2′,5′-and 3′,5′-phosphodiester linkages, may also be used to confirm the branched nature of the molecules according to the method of Ooi et al. (2001).

Basic Protocol 1
The synthesis of the branch synthon (S.3) from 5′-O-DMTr-N 6 -benzoyl-riboadenosine is extremely moisture sensitive. Thus, it is imperative that all solvents, reagents, and apparatuses (e.g., syringes, needles, reaction vessel) be anhydrous. The phosphitylating reagent (stored at −20°C) should be warmed to room temperature in a desiccator prior to use. If the isolated yield of S.3 is significantly lower than expected, verify that the phosphitylating chlorophosphoramidite is a clear viscous liquid, and that no crystalline material is present (this residue is indicative of hydrolysis).

Basic Protocol 2
There are two important considerations when synthesizing bNA by the convergent approach. Firstly, the yield of bNA is dependent upon the molar concentration of the branching bisphosphoramidite synthon S.3. Dilute solutions (0.03 M) of S.3 must be employed to maximize coupling with adjacent CPG-bound oligonucleotide chains (branching). If higher Current Protocols in Nucleic Acid Chemistry Supplement 9 4.14.29

Synthesis of Modified Oligonucleotides and Conjugates
concentrations are used, yields of branched products are reduced, and mainly extended isomeric side products are formed. The efficiency of the branching reaction can be assessed during synthesis by quantitation of the trityl cations released immediately before and after the branching reaction. Theoretically, 100% branch formation should give an apparent coupling yield of 50% since the trityl absorption following branching should be half of the previous absorption (during branching two nucleotide chains become joined to a single bisphosphoramidite synthon). Thus, trityl yields significantly greater or less than 50% indicate little branch formation (e.g., 80% apparent trityl yield indicates the formation of mainly the extended isomeric compounds). It is important to prepare the bisphosphoramidite stock solution (0.03 M) using at least 100 mg S.3. This ensures that (unavoidable) traces of moisture will not use up significant amounts of bisphosphoramidite during the branching step and reduce the overall yield of synthesis. The purity of the bisphosphoramidite can be assessed prior to use via TLC analysis or 31 P-NMR. If partial decomposition has occurred, or if the branching reactions during synthesis are poor, the compound should be rechromatographed as described (see Basic Protocol 1). Secondly, solid supports having a high degree of derivatization (>50 µmol/g) provide the best results for the synthesis of short bNA fragments (<7 nt). This is because highly substituted supports ensure the appropriate distance between the reactive 5′-OH end groups of the immobilized oligonucleotide chains. Supports with a nucleoside loading of 30 to 50 µmol/g give very good results for the synthesis of medium-size branched oligonucleotides.

Basic Protocol 3
The divergent approach requires synthesis of a linear oligonucleotide chain followed by backward synthesis from the 2′-hydroxyl of an internal ribonucleotide residue. This is carried out with commercially available inverted nucleoside 5′-phosphoramidites. Key to the success of this synthesis is the ability to cleave the 2′-O-silyl protecting group of the internal ribonucleotide residue and the use of an excess of the first inverted 5′-phosphoramidite reagent to force the branching reaction to a maximal extent. This is in sharp contrast to the convergent strategy, where branching requires a delicate control of reaction conditions. Incomplete removal of the 2′-O-TBDMS group from the RNA branch point (Fig. 4.14.8) results in the accumulation of the linear DNA-RNA-DNA precursor (S.12), which is easily detected by HPLC and PAGE. To ensure complete 2′-O-desilylation of the CPG-bound oligonucleotide, it is essential to use fresh TBAF reagent and to introduce the TBAF solution slowly to the synthesis column (via syringe), pushing and pulling the piston slightly to displace the CPG beads stuck at the base of the column. This allows all of the CPG-bound oligonucleotide to come in contact with the TBAF reagent. It is important not to expose the CPG-bound bNA to the TBAF reagent for >10 min, as prolonged treatment results in significant cleavage of the oligonucleotide from the solid support. After washing steps, the first inverted phosphoramidite is introduced as a 0.3 M acetonitrile solution (as opposed to 0.1 M for all other DNA 3′-and 5′-phosphoramidite couplings) and allowed to react with the CPGbound oligomer for 30 min (as opposed to 90 and 120 sec for all other DNA phosphoramidites).

Anticipated Results
The anticipated results of a convergent bNA synthesis are provided in Figures 4.14.6 and 4.14.7. Syntheses carried out on a 1-µmol scale will characteristically yield 50 to 80 A 260 units crude material, while the isolated yields (bNA) generally fall in the range of 5 to 20 A 260 units. bNAs that have been purified by anion-exchange HPLC are of high purity, usually >95% ( Fig. 4.14.10). In the case of divergent bNA synthesis, typical crude yields are in the range of 50 to 100 A 260 units. A standard gel analysis of bNA prepared by the divergent method is shown in Fig. 4.14.9. After HPLC or gel purification, 5 to 20 A 260 units of bNA are typically recovered from divergent syntheses (slightly higher yields are obtained with HPLC). As for convergent synthesis, purity is >95%.

Time Considerations
Provided that all reagents and materials required for each step are available, most of the procedures are simple and rapid. The synthesis of S.3 from 5′-DMTr-N 6 -benzoyl-riboadenosine requires 2 to 4 hr to complete, including the workup. The reaction should not be allowed to proceed overnight, as decomposition may occur. Column chromatography requires ∼1 to 2 hr including setup. Ideally, column purification should be conducted immediately following the phosphitylation reaction.
When the branching synthon S.3 and all the other phosphoramidite derivatives are ready to Supplement 9 Current Protocols in Nucleic Acid Chemistry 4.14.30 Solid-Phase Synthesis of Branched Oligonucleotides use, the time required to prepare, purify, and isolate a Y-shaped RNA or DNA via the convergent approach is 3 days and 4 to 6 days, respectively. The preparation of bNA via the divergent approach requires 3 days or more.