Nucleotidos 2 ppt

Clase anterior:-Reglas de Chargaff- Datos de difracción de rayos X- Estructura de la doble hélice- Componentes: pentosa, fosfato y base nitrogenada- Tipos de bases nitrogenadas- Tipos de enlaces en la doble hélice- Estructuras alternativas de la doble hélice

Nucleic acids can adopt different conformations.

* B-DNA is found at low salt concentrations. It is believed to be the native conformation occurring in chromatin.right-handed helix; sugar pucker: C2'-endo; number of nucleotides per pitch: 10; h: 3.38 Å; t: +36°.

* A-DNA is found in solutions with higher salt concentrations or with alcohol addedright-handed helix; sugar pucker: C3'-endo; number of nucleotides per pitch: 11; h: 2.56 Å; t: +32.7°.

* Z-DNA occurs for alternating poly(dG-dC) sequences in solutions with high salt concentrations or alcohol.left-handed helix; G: syn-conformation; sugar pucker: C3'-endo; C: anti-conformation, sugar pucker: C2' endo; number of nucleotides per pitch: 6x2; h:

3.7x2 Å; t= -30°x2

* RNA occurs (contrary to DNA) almost exclusively in the A-conformation (or in a related A'-form).

http://www.imb-jena.de/IMAGE_DNA_MODELS.html

dA dG

T dC

dA dG

T dC

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anti-conformation syn-conformation

In order for base-pairing to occur in a nucleic acid, the anti- conformation is

required.

dA dG

T dC





anti-conformation syn-conformation

In order for base-pairing to occur in a nucleic acid, the anti- conformation is

required.

In molecular biology, a wobble base pair is a non-Watson-Crick base pairing between two nucleotides in RNA molecules (codon-anticodon). The four main wobble base pairs are guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine (G-U, I-U, I-A and I-C). The thermodynamic stability of a wobble base pair is comparable to that of a Watson-Crick base pair.

A triple-stranded DNA is a structure of DNA in which three oligonucleotides wind around each other and form a triple helix. In this structure, one strand binds to a B-form DNA double helix through Hoogsteen or reversed Hoogsteen hydrogen bonds.

DNA triple hélice

The triplexes also form most readily within long sequences containing only pyrimidines or only purines in a given strand. Some triplex DNAs contain two pyrimidine strands and one purine strand; others contain two purine strands and one pyrimidine strand.

exotic DNA structure, known asH-DNA (Holiday junction) ,important during DNA recombination

A palindrome is a word, phrase, or sentencethat is spelled identically read either forward or backward;two examples are ROTATOR and NURSES RUN.

Secuencia palindrómica o palíndrome

DNA Replication

DNA replication is a semi-conservative process. One strand serves as the template for the second strand. DNA replication is initiated at a region on a chromosome called an origin of replication. An enzyme called DNA Helicase binds to the origin and unwinds the DNA in both directions from the origin.

Main types of RNAs

mRNAs

rRNAs

tRNAs

snRNAs

messenger RNAs, code for proteins.

ribosomal RNAs, components of the ribosome and catalyze protein synthesis.

transfer RNAs, carry specific amino acids to the elongating peptide chain during translation.

small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNAs.

RNA

Ribozimas

Otro “tipo” de ácido nucleico:

ssDNA

Funciones de los nucleótidos

Estructura del cAMP

Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues.

BrdU can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), substituting for thymidine during DNA replication.











Aciclovir- a guanine derivative, DNA polymerase inhibitor and antiviral



Nucleótidos modificados

Pseudouridine (Ψ) was discovered to be the most abundant posttranscriptionally modified nucleotide in cellular RNAs in the 1950s





DNA metiltransferasas







X

desaminación espontánea de citosina, se repara (DNA repair enzymes)

Xuracilo





desaminación espontánea de 5-metil citosina genera timina

(una mutación tipo transición)

Dideoxy Nucleotides

• Lack an -OH group at the 3-carbon position

• Cannot add another nucleoside at that position

• Prevent further DNA synthesis

http://web.utk.edu/~khughes/index.htm

Reversible denaturation and annealing of DNA

Efecto Hipocrómico / Hipercrómico

• Bases in DNA absorb light at 260 nm.

• The close proximity of the bases in double-stranded DNA quenches some of theabsorbance- hypochromic shift.

• When the two strands separate, this quenching disappears and the absorbancerises 30-40% hyperchromic shift.

Denaturation of DNA Causes Hyperchromism

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50 70 90

1.3

1.2

1.1

1.0

Temperature (C)

Re

lativ

e a

bso

rba

nce

(2

60

nm

)

Tm

Tm: temperatura a la cual el 50% del DNA es cadena doble

Depende de factores como:

Composición nucleotídicaEntorno (fuerza iónica, pH,…)Largo (complejidad) de las cadenas a hibridar

G+C Content Is Proportional to Tm

70 80 90 100

1.4

1.2

1.0Re

lativ

e a

bso

rba

nce

(2

60

nm

)

Temperature (C)

Pneumococcus(38%) G+C E. coli (52%)

S. Marcescens(58%)

M. Phlei(66%)

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2

3 X 105 moléculas de gene copia única corresponden a:

Tamaño genoma (pb)1 µg DNA humano ~3 X 109

10 ng DNA levadura ~1.6 X 107

1 ng DNA E. coli ~ 4 X 106

0.3 pg pBSSK ~2.9 X 103

Cation Neutralizes Phosphate and Elevates Tm

60 70 80 90 100 110

100

80

60

40

20

0

Tm (C)

G +

C (

%)

0.15 M NaCl0.015 M Na citrate

0.01 M phosphate0.001 M EDTA

Adapted from Garrett & Grisham (1999) Biochemistry (2e) p.372

3’

5’

5’

3’

ß-cianoethylphosphoramidite

1.Desprotección Consiste en eliminar mediante un tratamiento ligeramente ácido, al grupo Dimetoxitritilo (DMT). Este grupo protege al Hidroxilo 5' terminal de la base (nucleótido) correspondiente.

2.Acoplamiento Una vez libre el grupo hidroxilo, se adiciona el siguiente nucleótido, en forma de ß-cianoetilfosforamidito, correspondiente a la sequencia deseada.

3.Bloqueo (capping) Concluído el paso de acoplamiento, se bloquean todos los hidroxilos que no reaccionaron (menor al 2% del total) con la nueva base, esto mediante una reacción de acetilación.

4.Oxidación Finalmente, el grupo fosfito internucleotídico se oxida a un grupo fosfato estable.

Síntesis química de oligonucleótidos

TAREA:

¿Qué es una topoisomerasa?

¿Tipos de topoisomerasa y cuál son sus funciones?

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Nucleotidos 2 ppt