Nucleotides, Nucleic Acids PDF

Nucleotides, nucleic acids Lesson 1 Nucleotides Zsolt Fábián M.D., Ph.D., Dr. Habil.  Nucleotides, nucleic acids Identification of the genetic material – Griffith & Avery experiments Craig et al., M...

Nucleotides, nucleic acids Lesson 1 Nucleotides Zsolt Fábián M.D., Ph.D., Dr. Habil.  Nucleotides, nucleic acids Identification of the genetic material – Griffith & Avery experiments Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Identification of the genetic material – Hershey-Chase experiment Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotides deoxyadenosine 5′- monophosphate (dAMP)  Nucleic acids store and transmit genetic information  DNA and RNA are made from nucleotide building blocks  Nucleotides comprise a base, a sugar and phosphate(s)  Nucleotides have additional biological functions, such as energy storage (ATP) and molecular transport Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotides – the sugar components  Two types of sugar molecule are found in nucleotides  DNA contains deoxyribose  RNA contains ribose, and has an additional oxygen atom  In these sugars, the carbons are numbered 1′ to 5′ (said as “one prime” or “five prime”) Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotides – N-containing bases  DNA and RNA feature two types of base: purines and pyrimidines  Adenine and guanine are purines; cytosine, thymine and uracil are pyrimidines  DNA contains the bases adenine, guanine, cytosine and thymine  RNA does not have thymine, but uses uracil instead Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotides - bases  Bases are planar rings that are typically uncharged under physiological conditions  pKa values denote the pH at which half the molecules in a population are charged  Each base is joined to a sugar by a glycosidic bond between the 1′ carbon of the sugar and the first N of a pyrimidine or 9th N of a purine  Base plus sugar = nucleoside Nucleosides are named for bases: adenosine, guanosine, cytidine, thymidine and uridine  Nucleoside plus phosphate = nucleotide. Phosphate groups are linked to the 5′ carbon of the sugar Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotide nomenclature 1. Start with the nucleoside name Bases: Adenine, Guanine, Cytosine, Thymine, Uracil When in the corresponding Nucleoside: Adenosine, Guanosine, Cytidine, Thymidine, Uridine Note for the nucleoside names purines have –sine as the suffix pyrimidines have –dine as the suffix 2. If pentose is a deoxyribose, use the deoxy prefix e.g. deoxyadenosine, deoxyguanosine… 3. Indicate number of phosphates as 5' mono-, di- or tri- phosphates e.g. deoxyadenosine 5'-monophosphate deoxyguanosine 5'-triphosphate Nucleotides, nucleic acids Lesson 2 Ribonucleic acids Zsolt Fábián M.D., Ph.D., Dr. Habil.  Nucleotides, nucleic acids Nucleic acids  RNA is a polymer of nucleotides  Nucleotides are joined by a phosphodiester bond between the 3′ hydroxyl of one sugar and the phosphate attached to the 5′ hydroxyl of the next sugar  NA strands are thus directional – one end has an exposed 3′ hydroxyl, the other end has an exposed 5′ phosphate  By convention, NA sequences are written in the 5′ to 3′ direction  The sugars and phosphates form a repeating unit – the sugar-phosphate backbone  The backbone is always the same, but the attached bases vary RNA molecule Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA  RNA uses adenine, guanine, cytosine and uracil as bases  RNA has pentoses in the sugar- phosphate backbone that have hydroxyls at the 2’ carbons Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Modified RNAs (modRNA)  Many RNA molecules, especially directly functional RNAs, require chemical modification after synthesis known as post-transcriptional modification  Chemical modifications are usually permanent and are not regulatory  e.g. ~10% of the 75 nucleotides in tRNA are modified  Modifications are often conserved among species, reflecting their crucial functional roles Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Modified RNAs (modRNA)  Many RNA molecules, especially directly functional RNAs, require chemical modification after synthesis known as post-transcriptional modification  Chemical modifications are usually permanent and are not regulatory  e.g. ~10% of the 75 nucleotides in tRNA are modified  Modifications are often conserved among species, reflecting their crucial functional roles Credit to David M. Mauger et al. PNAS 2019;116:48:24075-24083 and Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Modified RNAs  In phosphorodiamidate morpholino oligomers  ribose (RNA) or deoxyribose (DNA) is replaced with 6-atom morpholine rings  ionic phosphodiester groups are replaced with uncharged non-ionic phosphorodiamidate groups  neutral and very resistant to degradation  Sarepta Therapeutics - Exondys 51 (Eteplirsen) Kole, et al., Nat Rev Drug Discov 11, 125–140 (2012)  Nucleotides, nucleic acids RNA conformation  The 2′ OH in ribose is important for RNA structure and function  The 2′ OH facilitates a reaction that can break phosphodiester bonds.  The 2′ OH means that RNA favors an A- type helix  RNA polymer has a relative low stability Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA folding RNAs often fold into complex, molecule-specific, three dimensional structures that includes:  Primary structure, which is the sequence of the bases in the RNA read in 5′ to 3′ direction  Secondary structures, that are short double- helical regions within the molecule  Tertiary structure, that is the arrangement of the double-helices and single stranded regions in the final configuration of the RNA Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Non-canonical RNA base pairs Wobble U-G pair Watson-Crick C-G pair  Another non-canonical pair is G-U, which has two hydrogen bonds (like A-U) - This is known as a “wobble” base pair Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Non-canonical RNA base pairs  RNA molecules often have some base pairs that are not Watson-Crick interactions  These non-canonical base pairs often feature chemically modified bases, such as methylation (addition of a CH3)  Pairing with modified bases can introduce structural differences such as kinks Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA folding  Tertiary RNA structure is formed when short double stranded helices interact with each other Base triple and with single-stranded regions interaction  Base pairs tend to stack – this leads to coaxial stacking, where helices stack with their axes aligned (as in tRNA)  Hydrogen bonding interactions, particularly involving the 2′ OH, are common - A base triple interaction has two Watson- Crick bases interacting with a third via an additional H-bond - In an A-minor motif, an adenosine inserts into the minor groove of a double-helical region, and is stabilized by H-bonds A-minor motif Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA conformations  An RNA double helix is short, usually 6- 8 base pairs, in A-helix formation  Helices are usually formed with Watson- Crick base pairing, in an antiparallel direction  If complementary sequences are close, a hairpin forms (the RNA folds back on itself)  In a hairpin, the double-stranded part is the “stem”, and the unpaired section is the “loop”  In tRNA, three of the double-helices are formed by hairpins, and one (in red) is formed from distant complementary regions Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA folding  RNA sequences can be searched computationally for potential base-pairing regions (shown in pink)  Because pairing regions are short, this approach can falsely identify interacting regions  Phylogenetic analysis (comparison of characteristics between species) helps identify true pairing regions, as these are more likely to be conserved during evolution  Differences that occur between species, yet conserve interactions, are known as covariations Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA folding  The phosphates in nucleic acids are negatively charged  When different parts of the backbone approach, they electrostatically repulse one another  RNA molecules bind large numbers of cations (like Mg2+, shown in red) to counteract this  This allows the RNA molecule to form a more compact and stable structure Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids RNA world  Proteins often require cofactors for function, many of which are RNA-derived, e.g., NAD+, providing further evidence for an RNA world  Each cofactor is used by many disparate proteins, suggesting early development and evolutionary conservation  Additional evidence includes the ability of RNA to fold in vitro and the discovery of RNA switches that regulate gene expression Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotides  Nucleotides and their derivatives also have important roles in other cellular functions Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleotide functions Cellular “energy currency” ATP and GTP are spent like you would spend a Nickle Mediators in cellular signaling mostly GDP & GTP Structural component of many enzyme cofactors and metabolic intermediates Eg. NAD+, FADH2, UDP-glucose  Nucleotides, nucleic acids Origin of nucleic acids Protein synthesis via the ribosome is an example of a conserved fundamental process that heavily involves RNA. The ribosome is made of RNA and protein, mRNA carries the information for the amino acid, and tRNA translates that information into protein sequence  It is not yet known how life evolved from mixtures of reactive molecules – “primordial soup” experiments readily generated amino acids but nucleic acid generation has thus far been difficult  LUCA likely depended on RNA, DNA, proteins and carbohydrates  Nucleic acids were likely involved in early life, due to their replicability and heritability  RNA can store biological information AND perform cellular functions and is thus thought to be the ancestral nucleic acid Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014) Nucleotides, nucleic acids Lesson 3 Deoxyribonucleic acids Zsolt Fábián M.D., Ph.D., Dr. Habil.  Nucleotides, nucleic acids Nucleic acids  DNA and RNA are polymers of nucleotides  Nucleotides are joined by a phosphodiester bond between the 3′ hydroxyl of one sugar and the phosphate attached to the 5′ hydroxyl of the next sugar  NA strands are thus directional – one end has an exposed 3′ hydroxyl, the other end has an exposed 5′ phosphate  By convention, NA sequences are written in the 5′ to 3′ direction  The sugars and phosphates form a repeating unit – the sugar-phosphate backbone  The backbone is always the same, but the attached bases vary DNA molecule Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Chargaff’s rule suggested base-pairing The amount of T always equals the amount of A, and the amount of C always equals the amount of G in double-stranded DNA – A=T – C=G The total amount of pyrimidine nucleotides (T+C) always equals the total amount of purine nucleotides (A+G) in double-stranded DNA – (A+G) = (C+T) – A+T+C+G = 100%  Nucleotides, nucleic acids Nucleic acids - DNA  Two DNA strands associate via non-covalent hydrogen bonds to form double-stranded DNA  Bases pair precisely with their complementary base: A pairs with T with 2 H-bonds C pairs with G with 3 H-bonds  These are called Watson-Crick base pairs  The sequence of one strand dictates the sequence of the other strand – thus the strands are complementary to one another  The two strands are antiparallel – the 5′ end of one strand pairs with the 3′ end of the other Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Identification of the genetic material – the Watson-Crick model Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Rosalind Elsie Franklin (25 July 1920 – 16 April 1958)  Nucleotides, nucleic acids Nobel prize in Physiology or Medicine - 1962 Francis Harry James Dewey Maurice Hugh Compton Crick Watson Frederick Watson & Crick, Nature (1953) 191:737-738  Nucleotides, nucleic acids DNA  The most energetically favorable formation of double-stranded DNA is for the two strands to wind around one another in a right-handed double helix  The hydrophobic bases cluster in the center, away from the aqueous cellular environment  The hydrophilic sugar phosphate backbone interacts favorably with water, and so is on the outside of the molecule  Base pairs form a stack on the interior of the helix. Van der Waals interactions between bases stabilize the interactions  This “base-stacking” arrangement is very energetically favorable, and is important for the stability of DNA Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids DNA  A-T and G-C base pairs have similar widths, so the helix diameter is ~20 Å  B-DNA is the predominant configuration. The helix repeats every 10.5 base pairs, and base pairs are 3.4 Å apart  In B-DNA, the helix forms a major groove (~13 Å) and a minor groove (~9 Å)  The shape and size of these grooves govern interactions with other molecules  The sequence of bases in DNA can influence structure: regions rich in A-T pairing tend to be more bendable Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids DNA  A-DNA is a right-handed helix, but has 11 base pairs per turn, making the grooves move evenly sized. The A conformation can be induced by DNA binding proteins.  Z-DNA, a left-handed helix, can result from methylation of cytosine, tortional stress, and high salt concentrations Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Pro- & eukaryotic DNA conformations Christiansen, et al., Journal of bacteriology (1973) 114:367-77 K. G. Murti and D. M. Prescott PNAS (1999) 96 (25):14436-14439  Nucleotides, nucleic acids DNA conformations  Closed circular DNA can be supercoiled  Supercoiled DNA is under tension and twists in on itself  Open, uncoiled circular DNA is said to be relaxed Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids DNA conformations  To induce supercoiling, a circular molecule is cut and held at one end while the other end is twisted  This changes the number of bases per turn  When the two ends are stuck back together (ligated) the DNA twists to restore the preferred number of bases per turn  This causes the DNA to wrap around itself in a coiled structure Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids DNA conformations  Supercoiling can be positive or negative, depending on the direction of the DNA twisting  Clockwise winding of the DNA, tending to separate the strands, leads to negative supercoiling  Twisting in the counterclockwise direction induces positive supercoiling  Linear pieces of DNA can also be supercoiled if one end is immobile  Supercoiling is released if one of the DNA strands is cut  Supercoiling can be toroidal or interwound Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Lesson 4 Physicochemical properties of nucleic acids Zsolt Fábián M.D., Ph.D., Dr. Habil.  Nucleotides, nucleic acids Physicochemical properties of DNA Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleic aids – Denaturation & Reannealing Denaturation - H-bonds between bases are broken, allowing strands to separate – Heat, high pH, low [salt], etc. – Leads to “melting” Reannealing (“renaturation” or “reassociation”) - separated strands reassociate or reanneal by specifically reforming the H-bonds in the base- pairs  Nucleotides, nucleic acids Hyperchromocity of DNA ssDNA A260 dsDNA Tm 20 30 40 50 60 70 80 90 100 T (C°) Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Nucleotides, nucleic acids Nucleic aids – Denaturation & Reannealing Nucleotides, nucleic acids Nucleic aids – Hybridization Hybridization Annealing of a single-strand of DNA to a complementary strand of a different DNA molecule Used in many molecular techniques – Allows detection of DNA molecules of a specific sequence in complex mixtures 100% complementarity is not necessary. By altering conditions, we can control the degree to which base-pair mismatches are tolerated Stringency = the ‘degree to which mismatches are tolerated’ in a hybridization reaction  Nucleotides, nucleic acids What are the names of these compounds?  Nucleotides, nucleic acids Please explain the figure DNA 1 DNA 2 Time Britten & Kohne, Repeated Sequences in DNA. Science, (1968) 161(3841), 529–540