Biochemistry, 4th Edition P38 docx

11.4 Can DNA Adopt Structures of Higher Complexity? 333 of hybridization is a measure of the sequence similarity or relatedness between the two species. Depending on the conditions of the experiment, about 25% of the DNA from a human forms hybrids with mouse DNA, implying that some of the nu- cleotide sequences (genes) in humans are very similar to those in mice (Figure 11.20). Mixed RNAϺDNA hybrids can be created in vitro if single-stranded DNA is allowed to anneal with RNA copies of itself, such as those formed when genes are transcribed into mRNA molecules. Nucleic acid hybridization is a commonly employed procedure in molecular biol- ogy. First, it can reveal evolutionary relationships. Second, it gives researchers the power to identify specific genes selectively against a vast background of irrelevant ge- netic material: An appropriately labeled oligonucleotide or polynucleotide, referred to as a probe, is constructed so that its sequence is complementary to a target gene. The probe specifically base pairs with the target gene, allowing identification and sub- sequent isolation of the gene. Also, the quantitative expression of genes (in terms of the amount of mRNA synthesized) can be assayed by hybridization experiments. 11.4 Can DNA Adopt Structures of Higher Complexity? DNA can adopt regular structures of higher complexity in several ways. For example, many DNA molecules are circular. Most, but not all, bacterial chromosomes are co- valently closed, circular DNA duplexes, as are most plasmid DNAs. Plasmids are nat- urally occurring, self-replicating, extrachromosomal DNA molecules found in bacte- ria; plasmids carry genes specifying novel metabolic capacities advantageous to the host bacterium. Various animal virus DNAs are circular as well. Supercoils Are One Kind of Structural Complexity in DNA In duplex DNA, the two strands are wound about each other once every 10 bp, that is, once every turn of the helix. Double-stranded circular DNA (or linear DNA du- plexes whose ends are not free to rotate) form supercoils if the strands are under- wound (negatively supercoiled) or overwound (positively supercoiled) (Figure 11.21). Un- derwound duplex DNA has fewer than the normal number of turns, whereas overwound DNA has more. DNA supercoiling is analogous to twisting or untwisting a two-stranded rope so that it is torsionally stressed. Negative supercoiling intro- Native DNA Heat Denatured DNA Nucleation (second-order) Slow Zippering (first-order) Fast Renatured DNA 1 2 FIGURE 11.19 Steps in the thermal denaturation and re- naturation of DNA.The nucleation phase of the reaction is a second-order process depending on sequence alignment of the two strands (1). This process takes place slowly because it takes time for complementary sequences to encounter one another in solution and then align themselves in register. Once the sequences are aligned, the strands zipper up quickly (2). Mix Denature, reanneal FIGURE 11.20 Solutions of human DNA (red) and mouse DNA (blue) are mixed and denatured,and the single strands are allowed to reanneal. About 25% of the human DNA strands form hybrid duplexes (one red and one blue strand) with mouse DNA. 334 Chapter 11 Structure of Nucleic Acids duces a torsional stress that favors unwinding of the right-handed B-DNA double he- lix, whereas positive supercoiling overwinds such a helix. Both forms of supercoil- ing compact the DNA so that it sediments faster upon ultracentrifugation or migrates more rapidly in an electrophoretic gel in comparison to relaxed DNA (DNA that is not supercoiled). Cellular DNA is almost always negatively supercoiled (underwound). Linking Number The basic parameter characterizing supercoiled DNA is the linking number (L). This is the number of times the two strands are intertwined, and provided both strands remain covalently intact, L cannot change. In a relaxed circular DNA du- plex of 400 bp, L is 40 (assuming 10 bp per turn in B-DNA). The linking number for relaxed DNA is usually taken as the reference parameter and is written as L 0 . L can be equated to the twist (T) and writhe (W ) of the duplex, where twist is the number of helical turns and writhe is the number of supercoils: L ϭ T ϩ W Figure 11.22 shows the values of T and W for a simple striped circular tube in var- ious supercoiled forms. In any closed, circular DNA duplex that is relaxed, W ϭ 0. A relaxed circular DNA of 400 bp has 40 helical turns, T ϭ L ϭ 40. This linking number can be changed only by breaking one or both strands of the DNA, winding them tighter or looser, and rejoining the ends. Enzymes capable of carrying out such reactions are called topoisomerases because they change the topological state of DNA. Topoisomerases fall into two basic classes: I and II. Topoisomerases of the I type cut one strand of a DNA double helix, pass the other strand through, and then rejoin the cut ends. Topoisomerase II enzymes cut both strands of a dsDNA, pass a region of the DNA duplex between the cut ends, and then rejoin the ends (Figure 11.23). Topoisomerases are important players in DNA replication (see Chapter 28). DNA Gyrase The bacterial enzyme DNA gyrase is a topoisomerase that introduces negative supercoils into DNA in the manner shown in Figure 11.23. Suppose DNA gyrase puts four negative supercoils into the 400-bp circular duplex, then W ϭϪ4, T remains the same, and L ϭ 36 (Figure 11.24). In actuality, the negative supercoils cause a torsional stress on the molecule, so T tends to decrease; that is, the helix be- comes a bit unwound, so base pairs are separated. The extreme would be that T would decrease by 4 and the supercoiling would be removed (T ϭ 36, L ϭ 36, and (a) (b) (c) Base of loop Interwound supercoil Toroidal spirals within supercoil FIGURE 11.21 Toroidal and interwound varieties of supercoiling. (a) The DNA is coiled in a spiral fashion about an imaginary toroid (yellow circle). (b) The DNA interwinds and wraps about itself. (c) Supercoils in long, linear DNA arranged into loops whose ends are restrained—a model for chromosomal DNA. (Adapted from Figures 6.1 and 6.2 in Callandine, C. R.,and Drew, H. R.,1992. Understanding DNA:The Molecule and How It Works. London: Academic Press.) 11.4 Can DNA Adopt Structures of Higher Complexity? 335 W ϭ 0). That is, negative supercoiling has the potential to cause localized unwind- ing of the DNA double helix so that single-stranded regions (or bubbles) are cre- ated (Figure 11.24). Usually the real situation is a compromise in which the nega- tive value of W is reduced, T decreases slightly, and these changes are distributed over the length of the circular duplex so that no localized unwinding of the helix ensues. Nevertheless, negative supercoiling makes it easier to separate the DNA strands and access the information encoded by the nucleotide sequence. Superhelix Density The difference between the linking number of a DNA and the linking number of its relaxed form is ⌬L ϭ (L Ϫ L 0 ). In our example with four negative supercoils, ⌬L ϭϪ4. The superhelix density or specific linking difference is defined as ⌬L/L 0 and is sometimes termed sigma, ␴. For our example, ␴ ϭϪ4/40, or Ϫ0.1. As a ratio, ␴ is a measure of supercoiling that is independent of length. Its sign reflects whether the supercoiling tends to unwind (negative ␴) or overwind (positive ␴) the helix. In other words, the superhelix density states the number of supercoils per 10 bp, which also is the same as the number of supercoils per B-DNA repeat. Circular DNA isolated from natural sources is always found in the underwound, negatively supercoiled state. Toroidal Supercoiled DNA Negatively supercoiled DNA can arrange into a toroidal state (Figure 11.25). The toroidal state of negatively supercoiled DNA is sta- bilized by wrapping around proteins that serve as spools for the DNA “ribbon.” This toroidal conformation of DNA is found in protein–DNA interactions that are the L = 0 L = +3 T = 0 W = 0 T = +3 W = 0 T = 0 W = +3 T = +1 W = +2 T = +2 W = +1 L = 0 L = – 3 T = 0 W = 0 T = – 3 W = 0 T = 0 W = – 3 T = – 1 W = – 2 T = – 2 W = 1 (1) (2) (3) (4) (5) (1) (2) (3) (4) (5) (a) Positive supercoiling (b) Negative supercoiling FIGURE 11.22 Supercoil topology for a simple circular tube with a single stripe along it. (Adapted from Figures 6.5 and 6.6 in Callandine, C. R.,and Drew, H. R.,1992. Understanding DNA:The Molecule and How It Works. London: Academic Press.) A B (+) node(–) node DNA is cut and a conformational change allows the DNA to pass through. Gyrase religates the DNA and then releases it. DNA loop AB AB AB AB A B A B (+) node(–) node (–) node(–) node A B ATP ADP + P i 1 2 3 4 FIGURE 11.23 A model for the action of bacterial DNA gyrase (topoisomerase II).The A-subunits cut the DNA duplex (1) and then hold onto the cut ends (2). Confor- mational changes in the enzyme allow an intact region of the DNA duplex to pass between the cut ends.The cut ends are religated (3), and the covalently complete DNA duplex is released from the enzyme.The circular DNA now contains two negative supercoils (4). 336 Chapter 11 Structure of Nucleic Acids basis of phenomena as diverse as chromosome structure (see Figure 11.27) and gene expression. 11.5 What Is the Structure of Eukaryotic Chromosomes? A typical human cell is 20 ␮m in diameter. Its genetic material consists of 23 pairs of ds- DNA molecules in the form of chromosomes, the average length of which is 3 ϫ 10 9 bp/23 or 1.3 ϫ 10 8 nucleotide pairs. At 0.34 nm/bp in B-DNA, this represents a DNA molecule 5 cm long. Together, these 46 dsDNA molecules amount to more than 2 m of DNA that must be packaged into a nucleus perhaps 5 ␮m in diameter! Clearly, the DNA must be condensed by a factor of more than 10 5 . The mechanisms by which this condensation is achieved are poorly understood at the present time, but it is clear that the first stage of this condensation is accomplished by neatly wrapping the DNA around protein spools called nucleosomes. The string of nucleosomes is then coiled to form a helical filament. Subsequent steps are less clear, but it is believed that this fila- ment is arranged in loops associated with the nuclear matrix, a skeleton or scaffold of proteins providing a structural framework within the nucleus (see following discussion). Nucleosomes Are the Fundamental Structural Unit in Chromatin The DNA in a eukaryotic cell nucleus during the interphase between cell divisions ex- ists as a nucleoprotein complex called chromatin. The proteins of chromatin fall into two classes: histones and nonhistone chromosomal proteins. Histones are abundant and play an important role in chromatin structure. In contrast, the nonhistone class is defined by a great variety of different proteins, all of which are involved in genetic reg- ulation; typically, there are only a few molecules of each per cell. Five distinct histones are known: H1, H2A, H2B, H3, and H4 (Table 11.2). All five are relatively small, posi- (a) Relaxed bp: L: T: W : 400 40 40 0 Gyrase + ATP (nicking and closing) bp: L: T: W : 400 36 40 –4 bp: L: T: W : 400 36 36 0 Strained: supertwisted (b) Strained: disrupted base pairs (c) FIGURE 11.24 A 400-bp circular DNA molecule in differ- ent topological states: (a) relaxed, (b) negative super- coils distributed over the entire length, and (c) negative supercoils creating a localized single-stranded region. (a) T = – 2, W = 0 (b) (c) T = 0, W = – 2 T = 0, W = – 2 Protein spool FIGURE 11.25 Supercoiled DNA in a toroidal form wraps readily around protein “spools.”A twisted segment of linear DNA with two negative supercoils (a) can collapse into a toroidal conformation if its ends are brought closer together (b). Wrapping the DNA toroid around a protein “spool” stabilizes this conformation (c). (Adapted from Figure 6.6 in Callandine, C. R., and Drew, H. R.,1992. Understanding DNA: The Molecule and How It Works. London: Academic Press.) Ratio of Histone Lysine to Arginine Size (kD) Copies per Nucleosome H1 59/3 21.2 1 (not in core) H2A 13/13 14.1 2 H2B 20/8 13.1 2 H3 13/17 15.1 2 H4 11/14 11.4 2 TABLE 11.2 Properties of Histones 11.5 What Is the Structure of Eukaryotic Chromosomes? 337 tively charged, arginine- or lysine-rich proteins that interact via ionic bonds with the negatively charged phosphate groups on the polynucleotide backbone. Pairs of his- tones H2A, H2B, H3, and H4 aggregate to form octameric core structures, and the DNA helix is wound about these core octamers, creating nucleosomes. If chromatin is swelled suddenly in water and prepared for viewing in the electron microscope, the nucleosomes are evident as “beads on a string,” dsDNA being the string. The structure of the histone octamer core wrapped with DNA has been solved by T. J. Richmond and collaborators (Figure 11.26). The core octamer has surface landmarks that guide the course of the DNA; 147 bp of B-DNA in a flat, left-handed superhelical conformation make 1.6 turns around the histone core (Figure 11.26), which itself is a protein superhelix consisting of a spiral array of the four histone dimers. Histone H1, a three-domain protein, organizes an additional 29–43 bp of DNA and links consecutive nucleosomes. Each complete nucleosome unit contains 176–190 bp of DNA. The N-terminal tails of histones H3 and H4 are accessible on the surface of the nucleosome. Lysine and serine residues in these tails can be covalently modified in myriad ways (lysines may be acetylated, methylated, or ubiquitinated; ser- ines may be phosphorylated). These modifications play an important role in chro- matin dynamics and gene expression (see Chapter 29). Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes A higher order of chromatin structure is created when the array of nucleosomes, in their characteristic beads-on-a-string motif, is wound in the fashion of a solenoid (Figure 11.27). One structure proposed for the resulting 30-nm fiber has a diameter of 33 nm and a height of 33 nm. It is formed by 22 nucleosomes arrayed helically. Cur- rent evidence indicates that this 30-nm filament then forms long DNA loops of variable length, each containing on average between 60,000 and 150,000 bp. Electron micro- scopic analysis of human chromosome 4 suggests that 18 such loops are then arranged radially about the circumference of a single turn to form a miniband unit of the chro- mosome. According to this model, approximately 10 6 of these minibands are arranged along a central axis in each of the chromatids of human chromosome 4 that form at mitosis (Figure 11.27). Despite intensive study, much about the higher-order structure of chromosomes remains to be discovered. (a) (b) FIGURE 11.26 The nucleosome core particle wrapped with 1.65 turns of DNA (147 bp). The DNA is shown as a blue and orange double helix.The four types of core histones are shown as different colors.(left) View down the axis of the nucleosome; (right) view perpendicular to the axis (pdb id ϭ 1AOI). (Adapted from Luger,K., et al., 1997. Crystal structure of the nucleosome core particle at 2.8 Å resolution.Nature 389:251–260. Photos courtesy of T. J. Richmond, ETH-Hönggerberg, Zurich, Switzerland.) 338 Chapter 11 Structure of Nucleic Acids SMC Proteins Establish Chromosome Organization and Mediate Chromosome Dynamics Although the details remain a mystery, we know that the process of chromatin or- ganization into chromosomes involves SMC proteins. SMC stands for structural maintenance of chromosomes. SMC proteins are members of the nonhistone chro- mosomal protein class. SMC proteins form a large superfamily of ATPases involved in higher-order chromosome organization and dynamics. SMC protein representa- tives are found in all forms of life—archaea, bacteria, and eukaryotes. Chromoso- mal dynamics includes chromosome condensation, sister chromatid cohesion, genetic recombination, and DNA repair, as well as other phenomena. SMC proteins have a characteristic five-domain organization, consisting of an N-terminal globular ATP-binding domain, a rodlike dimerization domain involved in coiled coil DNA double helix 2 nm “Beads on a string” chromatin form 10 nm Solenoid (six nucleosomes per turn) 30 nm Loops (50 turns per loop) ~ 0.25 ␮m Miniband (18 loops) Matrix 0.84 ␮m Chromosome (stacked minibands) 0.84 ␮m (a) (b) (c) (d) (e) FIGURE 11.27 A model for chromosome structure, hu- man chromosome 4. 11.6 Can Nucleic Acids Be Synthesized Chemically? 339 formation, a flexible hinge region, another rodlike and coiled coil–forming region, and finally a C-terminal globular domain termed DA for its DNA-binding and ATPase abilities (Figure 11.28). Five subgroups of SMC proteins are found in eu- karyotes, and functional SMC proteins are heterodimers. SMC2/SMC4 het- erodimers are essential for chromatin condensation as part of condensin com- plexes; SMC1/SMC3 heterodimers act in sister chromatid cohesion as part of cohesin complexes. Current models of SMC protein function suggest that V-shaped heterodimers bind to DNA through their DA domains and mediate chromosomal dynamics in an ATP-dependent manner. The flexible hinge region of each SMC subunit is located at the point of the V, and hinge-bending motions allow the DNA- binding parts of the two globular heads to move closer together, compacting the DNA into a higher-order structure (Figure 11.28). 11.6 Can Nucleic Acids Be Synthesized Chemically? Laboratory synthesis of oligonucleotide chains of defined sequence presents some of the same problems encountered in chemical synthesis of polypeptides (see Chapter 5). First, functional groups on the monomeric units (in this case, bases) are reactive under conditions of polymerization and therefore must be protected by blocking agents. Second, to generate the desired sequence, a phosphodiester bridge must be formed between the 3Ј-O of one nucleotide (B) and the 5Ј-O of the preceding one (A) in a way that precludes the unwanted bridging of the 3Ј-O of A with the 5Ј-O of B. Finally, recoveries at each step must be high so that overall yields in the multistep process are acceptable. As in peptide synthesis (see Chapter 5), orthogonal solid- phase methods are used to overcome some of these problems. Commercially available (a) SMC protein architecture SMC monomer (b) Chromatin condensation N–terminal ATP-binding domain DA domain Hinge region SMC2/SMC4 heterodimer DNA DNA Coiled coil domains SMC heterodimer FIGURE 11.28 SMC protein architecture and function. (a) SMC protein architecture. SMC proteins range from 115 to 165 kD in size.(b) SMC protein function.SMC pro- teins are reminiscent of motor proteins. Illustrated in (b) is a condensation of DNA into a coiled arrangement through SMC2/SMC4-mediated interactions. 340 Chapter 11 Structure of Nucleic Acids automated instruments, called DNA synthesizers or “gene machines,” are capable of carrying out the synthesis of oligonucleotides of 150 bases or more. Phosphoramidite Chemistry Is Used to Form Oligonucleotides from Nucleotides Phosphoramidite chemistry is currently the accepted method of oligonucleotide syn- thesis. The general strategy involves the sequential addition of nucleotide units as nucleoside phosphoramidite derivatives to a nucleoside covalently attached to the insol- uble resin. Excess reagents, starting materials, and side products are removed after each step by filtration. After the desired oligonucleotide has been formed, it is freed of all blocking groups, hydrolyzed from the resin, and purified by gel electrophore- sis. The four-step cycle is shown in Figure 11.29. Chemical synthesis takes place in the 3Ј→5Ј direction (the reverse of the biological polymerization direction). Genes Can Be Synthesized Chemically It is possible to synthesize genes using phosphoramidite chemistry (Table 11.3). Be- cause protein-coding genes are characteristically much larger than the 150-bp prac- tical limit on oligonucleotide synthesis, their synthesis involves joining a series of oligonucleotides to assemble the overall sequence. HUMAN BIOCHEMISTRY Telomeres and Tumors Eukaryotic chromosomes are linear. The ends of chromosomes have specialized structures known as telomeres. The telomeres of virtually all eukaryotic chromosomes consist of short, tandemly repeated nucleotide sequences at the ends of the chromosomal DNA. For example, the telomeres of human germline (sperm and egg) cells contain between 1000 and 1700 copies of the hexam- eric repeat TTAGGG (see accompanying figure). Telomeres con- tribute to the maintenance of chromosomal integrity by protect- ing against DNA degradation or rearrangement. Telomeres are added to the ends of chromosomal DNA by an RNA-containing enzyme known as telomerase (see Chapter 28). In human telom- erase, the ribonucleotide part is a 962-nucleotide-long RNA. Telomerase is an unusual DNA polymerase that was discovered in 1985 by Elizabeth Blackburn and Carol Greider of the University of California, San Francisco. However, most normal somatic cells lack telomerase. Consequently, upon every cycle of cell division when the cell replicates its DNA, about 50-nucleotide segments are lost from the end of each telomere. Thus, over time, the telomeres of somatic cells in animals become shorter and shorter, eventually leading to chromosome instability and cell death. This phenomenon has led some scientists to espouse a “telomere the- ory of aging” that implicates telomere shortening as the principal factor in cell, tissue, and even organism aging. Interestingly, can- cer cells appear “immortal” because they continue to reproduce indefinitely. A survey of 20 different tumor types by Geron Cor- poration of Menlo Park, California, revealed that all contained telomerase activity. 5'-CCTAACCCTAA 3'-GGGATTGGGATTGGGATT TTAGGGTTAGGGTTAGGG–3' AATCCC –5' TTAGGGTTAGGG AATCCC (a) 3'- 5'- 3' 5' Site of telomerase DNA polymerase function (b) Telomerase protein Telomerase RNA C AAUCCCAAUC ᮤ (a) Telomeres on human chromosomes.TTAGGG tandem repeats are attached to the 3Ј-ends of the DNA strands and are paired with the comple- mentary sequence 3Ј-AATCCC-5Ј on the other DNA strand.Thus, a G-rich region is created at the 3Ј-end of each DNA strand, and a C-rich region is created at the 5Ј-end of each DNA strand.Typically, at each end of the chromosome, the G-rich strand protrudes 12 to 16 nucleotides beyond its complementary C-rich strand. (b) The ribonucleic acid of human telomerase serves as the template for the DNA polymerase activity of telomerase. Nucleotides 46 to 56 of this RNA are CUAACCCUAAC and provide the template function for the telomerase-catalyzed addition of TTAGGG units to the 3Ј-end of a DNA strand. 11.7 What Are the Secondary and Tertiary Structures of RNA? 341 11.7 What Are the Secondary and Tertiary Structures of RNA? RNA molecules (see Chapter 10) are typically single-stranded. The course of a single-stranded RNA in three-dimensional space conceivably would have six de- grees of freedom per nucleotide, represented by rotation about each of the six sin- gle bonds along the sugar–phosphate backbone per nucleotide unit. (Rotation about the ␤-glycosidic bond creates a seventh degree of freedom in terms of the total conformational possibilities at each nucleotide.) Compare this situation with DNA, whose separated strands would obviously enjoy the same degrees of freedom. However, the double-stranded nature of DNA imposes great constraint on its con- formational possibilities. Compared to dsDNA, an RNA molecule has a much greater number of conformational possibilities. Intramolecular interactions and other stabilizing influences limit these possibilities, but the higher-order structure of RNA remains an area for fruitful scientific discovery. Adenine nucleotide NH 2 + C CH 3 C Cl N N N N R Benzoyl chloride HCl + NH N N N N R C O N-benzoyl adenine derivative N N N N H 2 N Guanine nucleotide + HC Cl Isobutyryl chloride HCl + N N N N R N-isobutyryl guanine derivative NH CO CH H 3 C CH 3 H O O O O R CH 3 (a) O CH 2 Base 1 O DMTr DMTr Detritylation by H + (trichloroacetic acid) O CH 2 Base 1 OR OH C OCH 3 CH 3 O Dimethoxytrityl (DMTr) OR Solid support (bead) 1 (b) BLOCKING GROUPS: FIGURE 11.29 Solid-phase oligonucleotide synthesis.The four-step cycle starts with the first base in nucleoside form attached by its 3Ј-OH group to an insoluble support. Its 5Ј-OH is blocked with a dimethoxytrityl (DMTr) group (a). If the base has reactive ONH 2 functions, as in A,G, or C,then N-benzoyl or N-isobutyryl derivatives are used to prevent their reaction (b). In step 1, the DMTr protecting group is removed by trichloroacetic acid treat- ment. Step 2 is the coupling step:The second base is added in the form of a nucleoside phosphoramidite deriv- ative whose 5Ј-OH bears a DMTr blocking group so it cannot polymerize with itself (c). continued Gene Size (bp) tRNA 126 ␣-Interferon 542 Secretin 81 ␥-Interferon 453 Rhodopsin 1057 Proenkephalin 77 Connective tissue activating peptide III 280 Lysozyme 385 Tissue plasminogen activator 1610 c-Ha-ras 576 RNase T1 324 Cytochrome b 5 330 Bovine intestinal Ca-binding protein 298 Hirudin 226 RNase A 375 TABLE 11.3 Some Chemically Synthesized Genes 342 Chapter 11 Structure of Nucleic Acids Although single-stranded, RNA molecules are rich in double-stranded regions that form when complementary sequences within the chain come together and join via intrastrand base pairing. These interactions create hairpin stem-loop structures, in which the base-paired regions form the stem and the unpaired regions between base pairs are the loop, as in Figures 11.30 and 11.31. Paired regions of RNA can- not form B-DNA-type double helices because the RNA 2Ј-OH groups are a steric hindrance to this conformation. Instead, these paired regions adopt a conforma- tion similar to the A-form of DNA, having about 11 bp per turn, with the bases + N HC HC OR O CH 2 Base 2 O O P H 3 CO CH 3 CH 3 H 3 C O CH 2 Base 1 OH OR HN CH 3 H 3 C HC H H N N N N OCH 3 O CH 2 Base 2 O O P O CH 2 Base 1 O Phosphoramidite derivative of nucleotides 2 OR OCH 3 O CH 2 Base 2 O O P O CH 2 Base 1 O O I 2 ; H 2 O oxidation of trivalent phosphorus Next nucleotide added following detritylation as in step 1. Cycle repeated to synthesize oligonucleotide of desired sequence and length. Cleavage of oligonucleotide from solid support and removal of N-benzoyl and N-isobutyryl blocking groups. NH 4 OH treatment Desired product Phosphite-linked bases (dinucleotide) CH 3 CH 3 HC CH 3 Phosphate-linked bases (dinucleotide) Catalyzed by weak acid tetrazole 2 Capping 3 4 (c) DMTr DMTr DMTr FIGURE 11.29 continued In step 2, the presence of a weak acid, such as tetrazole, activates the phospho- ramidite, and it rapidly reacts with the free 5Ј-OH of N-1, forming a dinucleotide linked by a phosphite group. Unreacted free 5Ј-OHs of N-1 are blocked from further participation in the polymerization process by acetyla- tion with acetic anhydride in step 3, referred to as capping. In step 4, the phosphite linkage between N-1 and N-2 is oxidized by aqueous iodine (I 2 ) to form the desired more stable phosphate group. Subsequent cycles add successive residues to the resin-immobilized chain.When the chain is complete, it is cleaved from the sup- port with NH 4 OH, which also removes the N-benzoyl– and N-isobutyryl–protecting groups from the amino functions on the A, G, and C residues.