Nucleic Acid Structure and Function

Nucleic Acid Structure and Function Nucleotides and Nucleosides o Building blocks of DNA o Nucleotides consist of: o A nitrogenous base o A ribose or deoxyribose sugar o One or more phosphoryl groups o Nucleosides lack phosphates Nucleotide bases are derivatives of pyrimidine and purine o There are five common bases in DNA and RNA o Thymine is found in DNA only o Uracil is found in RNA Only Nucleotide sugars are five-membered sugars o o o o o 5-membered cyclic sugars are known as furanoses Ribose is found in RNA Deoxyribose is found in DNA The difference is at the 2’ position Note the prime symbol in numbering carbons: allows us to distinguish between the sugar atoms and those in the nitrogenous base Phosphates in nucleotides can be found at the 5’-position or the 3’-position o By modifying the building blocks (bases, phosphates and sugars), we can generate diverse nucleotides/nucleosides Tautomeric forms of bases o Tautomer’s: structures that differ in locations of hydrogens o Predominant: amino (A,C), keto (G,T) forms o Rare: imino (A,C), enol (G,T) forms o Different tautomeric forms of the nitrogenous bases have different hydrogen bonding capacity What positions are involved in sugar attachment to base? o Bond between sugar and base is called a glycosidic bond Syn vs anti nucleotides o o o o Rotation can occur around the glycosidic bond Syn nucleotides: base above the furanose ring Anti nucleotides: base away from furanose ring In free nucleotides, the glycosidic bond rotates freely Nucleic acid polymers o Polymers form when phosphodiester bonds connect nucleotide monomers o Why is it called phosphodiester? o CO2R o Ends of DNA polymer strand are 5’ and 3’ o 5’-end of a nucleic acid: free phosphate group o 3’-end of a nucleic acid: free hydroxyl group Double helical DNA structure o o o o DNA adopts a double helix structure with a major and minor groove The DNA helix has a sugar-phosphate backbone The DNA strands are anti-parallel The nitrogenous bases are located on the interior of the helix. They are stacked horizontally to maximize non-covalent interactions 3 different types of DNA helices o B-form o First model identified (Watson-Crick-Franklin) o Most common form of DNA o A-form o Right-handed o More vertically compressed o Similar major and minor grooves o Found in RNA o Z-form o Left-handed o Transient, induced during transcription How is double stranded DNA stabilized? o Hydrogen bonding between strands (base pairing) o Pi stacking within a strand (base stacking) o Base pairing is facilitated by anti-parallel strands Base stacking o Pi stacking: orbital overlap between pi bonds of the aromatic rings o Attractive, non-covalent interaction o Driven by the hydrophobic effect Major and minor grooves o Depending on the base pair, there are different H bond donor or acceptor patterns o Atoms in the major and minor groove act as H-bond donors and acceptors o Important for DNA sequence recognition by proteins Non-canonical base pairs o W-C-F are canonical base pairs o But there are many types of base pairs RNA forms more diverse structures o RNA can fold back on itself to adopt diverse secondary structures o The 2’ OH of RNA will not fit into a B-form helix, so RNA adopts an A-form helix o Some RNAs can act as enzymes because of their structural stability o These are called ribozymes RNA structure is important for its function o tRNA (transfer RNA) o Important for protein synthesis o Need to maintain specific fold for function o Binding amino acids o Interacting with ribosome, enzymes, etc. RNA is less stable than DNA o Free hydroxyls in the environment induce deprotonation of 2’-OH o Intramolecular nucleophilic attack on adjacent phosphate o Cleavage of phosphodiester backbone Nucleic Acid Methods Nucleic acid methods o These methods provide the ability to manipulate, modify or determine the sequence of DNA/RNA o Cloning o PCR o Sanger sequencing o Gene microarrays o RNA-seq (next generation transcriptome using RNA sequencing) o CRISPR Cloning o Technique to make many identical copies of DNA o Plasmids are used for cloning o Circular pieces of DNA o Able to replicate independently from the host o Multiple cloning sequence: used to insert foreign DNA into plasmid o Performed using restriction enzymes (sequence specific endonucleases) o Produces recombinant DNA (produced in laboratory, combined from multiple sources) Restriction enzymes/ endonucleases o o o o o Cleave DNA by recognizing specific sequences Type II restriction enzymes cleave within the recognition sequence Recognize palindromic DNA sequences Cleave double-stranded DNA to generate 5’ overhangs, 3’ overhangs or blunt ends Overhangs are called sticky ends Incorporating DNA into plasmids o Recombinant DNA technology o Digest gene of interest and plasmid with the same restriction enzyme o Anneal both to promote hydrogen bonding between complementary sequences on sticky ends o Use DNA ligase to covalently join fragments. Ligase catalyzes formation of phosphodiester bond DNA polymerases o DNA polymerases extend a DNA strand during replication o Requirements o Template strand—which will be duplicated o Primer to be extended. Requires a 3’ OH for extension o dNTPs (deoxynucleotide triphosphates)—which are added to the growing strand ▪ CTP, GTP, ATP, TTP o During reaction, a new phosphodiester bond is formed, a diphosphate is released ▪ Nucleophile: 3’ OH ▪ Leaving group: diphosphate group How does primer bond? o Primer: short single-stranded nucleic acid sequence o Bind in an anti-parallel fashion o Are extended on the 3’ end Using RNA in cloning o Reverse transcription: synthesis of DNA from RNA o E.g. to identify all genes actively transcribed from mRNA in a cell o Convert mRNA to DNA using reverse transcriptase o RNA-dependent DNA polymerases o DNA is known as complementary DNA (cDNA) o Creates a DNA—RNA hybrid o Ribonuclease H is added to cleave RNA o cDNA becomes the new template Polymerase Chain Reaction (PCR) o Used for DNA amplification: generating multiple copies of a specific DNA target segment PCR cycle o PCR happens in 3 temperature phases o Denaturation at high temperature to separate DNA strands o Annealing at lower temperature to allow primers to anneal to each strand o Extension of primer and DNA synthesis at higher temperature PCR application o Each cycle doubles the number of DNA molecules fed into it (exponential amplification) o Commercial applications of PCR: o Diagnostics e.g. genetic testing, disease testing o Forensics o Agriculture e.g. GMO testing Sanger sequencing o To determine the sequence of a DNA molecule known as chain termination method o Requirements: o Polymerase o Template strand (extend growing chain) o Primer to be extended. Requires a 3’OH for extension o dNTPs (deoxynucleotide triphosphates) o ddNTPs (dideoxynucleotide triphosphates) Agarose gel electrophoresis o Agarose electrophoresis separates DNA based on size o The negative charge on sugar-phosphate backbone of DNA pulls it through the agarose matrix o Smallest DNA molecules run farthest from the top o DNA gels are stained with fluorescent intercalator dyes, such as ethidium bromide (EtBr) o An intercalator is a flat aromatic molecule that slips in between DNA bases in doublehelical DNA o EtBr is fluorescent when bound to DNA but quenched when it is free in solution Monosaccharides Carbohydrates are diverse in size, structure and function o Carbohydrates have a large range of molecular weight, from glyceraldehyde (90 Da) to amylopectin (> 200,000 kDa) o Functions include: o Energy o Structure o Information transfer o Carbohydrates can be linked to: o Proteins (glycoproteins, proteoglycans) o Lipids (glycolipids) Classification based on functional group o 2 broad classes of monosaccharides o Aldoses: aldehyde functional group. Carbonyl C is the first in the chain o Ketoses: ketone functional group. Carbonyl C is the second in the chain Number of carbons o Sugars can be classified by number of carbons o 3 carbons: trioses o 4 carbons: tetraoses o 5 carbons: pentoses o 6 carbons: hexoses o Stereoisomers have the same atom connectivity but different arrangements of substituents Enantiomers o Chiral compounds lacking a plane of symmetry o Mirror images that cannot be superimposed o D-isomer: OH on the right o L-isomer: OH on the left o If multiple chiral centers, D or L, is assigned to C farthest away from carbonyl C Diastereomers o Diastereomers are stereoisomers that are not mirror images o Differ at one or more—but not all—chiral centers o Diastereomers have different physical properties Epimers o Epimers differ only in the configuration around 1 C atom o Galactose is an epimer of glucose at C4 (C4-epimers) o Mannose is an epimer of glucose at C2 (C2-epimers) Cyclic monosaccharides form spontaneously o o o o More stable, more prevalent that open chain form Involves reaction of an alcohol with aldehyde or ketone group of sugar Reaction between aldehyde and alcohol: hemiacetal Reaction between ketone and alcohol: hemiketal Intramolecular cyclization of sugars o An OH on the carbon backbone attacks the carbonyl to produce a hemiacetal or hemiketal structure o A new chiral carbon is formed by the former C=O. Known as the anomeric carbon o The OH group at the anomeric C can have 2 possible configurations How to identify anomeric carbon o Locate O in the ring o Look at C on either side o Anomeric C has -OH directly attached (unmodified sugar) Converting from Fisher to Haworth projections o The OH must be oriented properly to attack the carbonyl o Must rotate around C4-C5 bond to line up the -OH for C1 C=O attack o The processes causes CH2OH to point up out of the ring Cyclic sugars are usually 5- or 6-membered o o o o o 5 membered ring is a furanose 6 membered ring is a pyranose The identity of the attacking -OH group determines the ring size Glucopyranose is formed by carbonyl attack by the OH on C5 Glucofuranose is formed by carbonyl attack by the OH from C4 Isomers of glucose o In solution, pyranose forms of glucose are more stable, thus more prevalent o They exist in equilibrium between alpha and beta forms o Very small amounts exist in open0chain and furanose forms Carbohydrates Monosaccharides can be modified o o o o o o o Oxidation of the aldehyde in a sugar yields an aldonic acid Oxidation of a primary OH yields a uronic acid Reduction of aldehyde in aldose sugar gives an alditol or sugar alcohol Alditols only contain hydroxyl groups, no carbonyl groups Reduction of an OH yields a deoxy sugar Amine substitution of OH yields an amino sugar The amino group can be acetylated to yield N-acetyl sugar: NAC Glycosidic Bond o Glycoside: a sugar joined to another non-sugar molecule by a glycosidic bond o Glycosidic bond covalently joins sugar to another molecule through the anomeric carbon o Formed by reaction between hemiacetal/hemiketal and a nucleophile to make an o O-glycoside (R-OH) o N-glycoside (R-NH2) Disaccharides o 1st sugar always uses anomeric carbon, 2nd sugar can use any carbon o Two sugars linked by a glycosidic bond o Can form in diverse ways o Monosaccharides o Anomers o Carbons in glycosidic linkage Naming glycosidic linkages o o o o o Identify anomeric C involved in glycosidic linkage Determine the number of that carbon Assign C as alpha or beta Look at other side of glycosidic bond, determine number of carbon on second sugar If the second sugar has a free anomeric carbon, determine overall alpha or beta form Reducing and non-reducing ends o o o o Disaccharides can have a reducing and nonreducing end Reducing end: end with a free anomeric carbon Non-reducing end: end without a free anomeric carbon Sugar is nonreducing if there is no free anomeric carbon Homopolysaccharides o Hundred of repeating sugar units o E.g. cellulose and amylose differ only in the alpha/beta orientation of linkage o Small structural difference = big consequences for the structure and function of these two polysaccharides o Loosely packed, helical structure o Function: energy storage o Tightly packed, dense, sheet-like structure o Function: structure, e.g. cell walls Polysaccharides o Polysaccharides can be disaccharide heteropolymers o E.g. chitin o Polysaccharides can also be branched o E.g. amylopectin Glycoproteins o Sugars are conjugated to proteins to generate proteoglycans and glycoproteins o N-linked glycoproteins link a sugar to the -NH2 of an Asn o O-linked glycoproteins link a sugar to the -OH of a Ser/Thr Monosaccharide symbols o Most glycoproteins use the same monosaccharide building blocks o For ease of representation, these can be depicted as colored symbols Glycoproteins and viruses o Many proteins in viruses (viral proteins) are glycosylated, promoting their parasitic activity o E.g. spoke protein from coronavirus is glycosylated Lipids 1 Functional categories of lipids o Lipid: any molecule more soluble in organic solvent than in water o No defined structure o Three important roles: o Energy storage o Cell membrane o Signaling Fatty acids o o o o o Fatty acids: carboxylic acids with hydrocarbon chains (4 to 36 carbons in length) Most lipids are derived from fatty acids Saturated FAs contain 0 double bonds in alkyl chain Unsaturated FAs contain double bonds in alkyl chain Polyunsaturated FAs contain more than 1 double bond in alkyl chain Melting temperatures o Saturated chains are extended, allowing tight packing and more interactions o Unsaturated chains are kinked, allowing weaker/looser packing and fewer interactions Fatty acid derived lipids o Fatty acids can exits alone or linked to a backbone (ester/amide linkage) o Two backbone molecules: o Glycerol (glycerolipids) o Sphingosine (sphingolipids) o Each has positions to attach fatty acids or polar head groups Fatty acid derived lipids – glycerolipids o o o o Glycerol is the backbone for glycerolipids 3-carbon carbohydrate with -OH on each C Fatty acids can be conjugated to all three carbons, or to 2 A polar head group occupies the their position in the latter case Fatty acid derived lipids—sphingolipids o Sphingosine is the backbone molecule for sphingolipids o An 18C amino alcohol with an unsaturated hydrocarbon chain o Headgroup at position 1 and fatty acid conjugated at position 2 Classification of FA-derive lipids o Lipids can be classified based on the o Backbone (glycerol, sphingosine) o Type of headgroup, and o Linkage between the two o Common head groups: o Phosphate/phosphate-linker: connected to bb through a phosphodiester bond o Sugars/sugar-based: connected to bb through a glycosidic bond Naming glycerophospholipids o First part of name described the fatty acid chains o Second part of name describes the head group and linkage Phospholipases o Enzymes that hydrolyze glycerophospholipids o These reactions are important for recycling and lipid signaling