Structure Determination

2. Structure Determination High resolution techniques - EM, NMR, X-ray Biochemical techniques - 1. Use chemicals that react with DNA so that cleavage occurs at all bases (not sequence selective) so as to create a DNA/RNA cleavage ladder; alterations in uniform cleavage ladder can be used to footprint DNA/RNA 2. Basic strategy to probe DNA/RNA conformation with chemicals is to generate some type of conformation-related DNA/RNA modification that can be converted into a strand break which can then be mapped Reagents react with one specific base Specificity with respect to: specific base specific base when it is in a unique conformation chemical not specific but method used to generate strand break is

3. Structure Determination Hydrolysis reaction: Used by nucleases (ribo- and deoxy-) Used by catalytic RNAs

4. Structure Determination Nucleases

5. Structure Determination Nucleases -restriction endonucleases Sequence-specific 1. Compatible cohesive ends EcoRI restriction endonuclease GAATTC CTTAAG G AATTC CTTAA G 2. Blunt ends HaeIII restriction endonuclease GGCC CCGG GG CC CC GG

6. Structure Determination Primary Maxam-Gilbert chemical sequencing of DNA uses base-specific chemical reactions to break chain at A, G, C or T G + A  piperidine in acid C + T  hydrazine and piperidine in base G  DMS and piperidine in base C  hydrazine, 1.5 M NaCl and piperidine in base 1. A base-specific rxn either cleaves aromatic ring of base or breaks glycosidic bond 2. The first step destabilizes deoxyribose ring and allows attack by piperidine with subsequent ring cleavage 3. Cleavage of phosphodiester chain 4. Conversion of phosphodiester to phosphomonoester

7. Structure Determination Primary Maxam-Gilbert chemical sequencing of DNA G + A  piperidine in acid

8. Structure Determination Primary Maxam-Gilbert chemical sequencing of DNA G  DMS and piperidine in base

9. Structure Determination Primary Maxam-Gilbert chemical sequencing of DNA C + T  hydrazine and piperidine in base C  hydrazine, 1.5 M NaCl and piperidine in base

10. Structure Determination - Primary Sanger dideoxy sequencing of DNA 5’-AATCGACTAGTACTAGTCTAGCTA-3’ 3’-TTAGCTGATCATGATCAGATCGAT-5’ Denature, 95 ˚C 5’-AATCGACTAGTACTAGTCTAGCTA-3’ + 3’-TTAGCTGATCATGATCAGATCGAT-5’ Anneal radiolabeled primer **5’-AATCGACTAGT 3’-TTAGCTGATCATGATCAGATCGAT-5’ Extend with DNA polymerase dCTP, dATP, dGTP, dTTP ddCTP ddTTP ddATP ddGTP **5’-AATCGACTAGTAddC **5’-AATCGACTAGTACddT **5’-AATCGACTAGTACTAGTddC **5’-AATCGACTAGTACTAGddT **5’-AATCGACTAGTACTAGTCTAGddC **5’-AATCGACTAGTACTAGTCddT **5’-AATCGACTAGTACTAGTCTAGCddT **5’-AATCGACTAGTACTAddG **5’-AATCGACTAGTddA **5’-AATCGACTAGTACTAGTCTAddG **5’-AATCGACTAGTACTddA **5’-AATCGACTAGTACTAGTCTddA **5’-AATCGACTAGTACTAGTCTAGCTddA Denature Gel electrophoresis

11. Structure Determination - Primary Sanger dideoxy sequencing of DNA 2’ 3’

12. Structure Determination - Primary Sanger dideoxy sequencing of DNA

13. Structure Determination - Primary Determination of RNA sequence 1. First convert RNA to DNA using reverse transcriptase 2. Peattie Chemical sequencing of RNA (similar to Maxam-Gilbert) uses base-specific chemical reactions to break chain at A, G, C or U Uses aniline at pH 4.6 to cleave RNA at site of modification G  DMS and aniline U  hydrazine and aniline A > G  DEPC and aniline C > U  hydrazine and 3 M NaCl and aniline 3. Ribonuclease sequencing RNases are base-specific, cut on the 3’ side of nucleotide label RNA at 5’ or 3’ end, add RNase, PAGE, also run OH- hydrolysis ladder

14. Structure Determination - Primary 2. Peattie Chemical sequencing of RNA (similar to M-G) G  DMS and aniline U  hydrazine and aniline A > G  DEPC and aniline C > U  hydrazine and 3 M NaCl and aniline

15. Structure Determination - Secondary Probe reactive sites with chemicals - if ds or ss only some sites can be modified For RNA and DNA covalent rxns can reveal which bases are (i) single-stranded (unbonded and usually most reactive), (ii) which are in base-pairs (hydrogen bonded) and (iii) which are maybe involved in 3˚ interactions Precise locations of binding of proteins & drug molecules can be determined by differences in reactivity of bound and naked DNA Experiments: Radiolabel DNA or RNA At 5’-end with [-32P] ATP and polynucleotide kinase At 3’-end with RNA ligase and radioactive pNp Chemical reactivity determined by: 1. Chemical cleavage of an end-labeled chain at site of modification refer to M-G seq and use of aniline (RNA) and piperidine (DNA) 2. Chain termination by reverse transcriptase at this site RT makes a complementary copy of RNA or DNA single strands, but it stops or pauses at modified bases or at the end of the strand where cleavage occurred. Length of copy identifies location of modified base (seq rxns done in parallel to provide exact position)

16. Structure Determination - Secondary Probe reactive sites in DNA/RNA with chemicals

17. Structure Determination - Secondary Probe reactive sites with chemicals All nitrogens and oxygens in base, sugar & phosphate can be alkylated Rxns with A and C << in dsDNA

18. Structure Determination - Secondary Probe reactive sites with chemicals - all these monitor W-C base pairing ability Good probes of duplex formation

19. Structure Determination - Secondary Probe reactive sites with chemicals - react faster in ss regions

20. Structure Determination - Secondary RNA - use all reagents in Table 3-2 Base specific nucleases (Table 3-1) are all more reactive on ssRNA than on dsRNA S1 hydrolyzes ssRNA or ssDNA but it is inhibited by ds structure Ex: in tRNA only anticodon loop is cut RNase V1 is specific for dsRNA and stacked ssRNA LIMITS of Nucleases - size of enzymes limit accessibility to site OH- cleavage nonspecific cleavage of backbone

21. Structure Determination - Secondary DNA - mostly duplex but do get ssDNA at cruciforms, triplex, junctions Can also use chemical reagents to determine where proteins bind - FOOTPRINT Cruciforms - ss loops hyperactive to ss-selective reagents bromoacetaldehyde, kethoxal, OsO4, bisulfite Triplex - induced in polypurine rich regions by (-) supercoiling and low pH, in H-DNA ss released and becomes hyperactive to ss-selective reagents, also G in triplex is protected from alkylation at N7 by DMS Nucleases - S1 hydrolyzes ssDNA/RNA FeII(EDTA) is an effective cleaving agent because hydroxyl radicals are oxidative agents that form when ferrous ions are in the presence of O2 (H2O2) and a reducing agent (ascorbate) •OH cleaves the sugar-phosphate backbone of DNA FeII(EDTA) complexes have been applied to DNA and to probing the solvent accessibility of RNA

22. Structure Determination - Secondary

23. Structure Determination - Secondary DNA Fe(EDTA)2- + H2O2 + ascorbate Fe2+ H2O2 •OH •OH •OH OH- •OH •OH Fe3+ O P O- O Base O O •OH O P O- O O O- O P O- O Base falls off P O- O O O-

24. Structure Determination - Secondary DNA Methidiumpropyl-EDTA + Fe (II) + O2 This reagent binds via intercalation, with little sequence discrimination and delivers oxidizing agent to the DNA

25. Structure Determination - Secondary NAIM - Nucleotide Analog Interference Mapping • Allows simultaneous, yet individual, identification of important functional groups within a large RNA • Nucleotide analogs are randomly incorporated into RNA during in vitro transcription • Selective assay for RNA function to separate active from inactive ribozymes • Analogs have a chemical tag - phosphorothioate linkage - to identify site of incorporation

26. Structure Determination - Secondary NAIM - Nucleotide Analog Interference Mapping

27. Secondary versus tertiary structure • Secondary structure can be drawn in 2D without any crossing lines • Tertiary interactions cross the lines of the secondary structure (chord crossing)

28. Structure Determination - Tertiary 1. use all chemicals/enzymes used for secondary structure because tertiary contacts can protect regions from chemical modification or nuclease cleavage 2. Chemical crosslinking - using psoralen and/or UV light reagents have 2 reactive groups that can be used to covalently link nucleotides that are far apart in sequence; determine location by EM or partial hydrolysis Combine crosslinking data with chemical probing and phylogeny to get a precise 3-dimensional structure

29. Structure Determination - Tertiary Chemical crosslinking - using psoralen Used with many nucleic acids, such as 16S rRNA and viral DNA 1. Intercalation of psoralen (intercalators are planar and stack as bases) 2. Psoralen absorbs a photon ( = 300-400 nm) 3. C=C on psoralen reacts by cycloaddition to C5=C6 of pyrimidine 4. Psoralen can then form a second crosslink to another pyrimidine T, U >> C

30. Structure Determination - Tertiary Chemical crosslinking - using UV light Use 200-300 nm light to cause crosslinks in DNA Formation of pyrimidine dimers linked by a cyclobutyl ring across C5=C6 Advantage because no chemical agent has to be added to solution

31. Structure Determination - Secondary Intercalation - planar aromatic molecules binding to DNA

32. Structure Determination - Secondary Intercalation - separation of base pairs with a lengthening of double helix and a decrease in helical twist (unwinding)

33. Structure Determination - Secondary Intercalator - ditercalinium is a bisintercalator (rigid chain) Used as an anticancer drug because it induces DNA repair in many spots on the cell’s DNA (nonspecific, noncovalent binding) which eventually causes the cell to die because of extensive repair-induced DNA degradation

34. 23S rRNA (then & now)