Nucleotidos 2

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 added
right-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
anti-conformation syn-conformation
In order for base-pairing

to occur in a nucleic
QuickTime™ and a
TIFF (Uncompressed) decompressor
QuickTime™ and a
are needed to see this picture.
acid, the anti-
conformation is
required.
dA dG
T dC
anti-conformation syn-conformation
In order for base-pairing

to occur in a nucleic
QuickTime™ and a
QuickTime™ and a
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.
DNA triple hélice
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.
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 as
H-DNA (Holiday junction)
,important during DNA
recombination
Secuencia palindrómica o palíndrome
A palindrome is a word, phrase, or sentence
that is spelled identically read either forward or backward;
two examples are ROTATOR and NURSES RUN.
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.
Otro “tipo” de ácido nucleico:
RNA
Main types of RNAs
mRNAs messenger RNAs, code for proteins.
rRNAs ribosomal RNAs, components of the ribosome and catalyze

protein synthesis.
tRNAs
transfer RNAs, carry specific amino acids to the elongating
peptide chain during translation.
snRNAs small nuclear RNAs, function in a variety of nuclear processes,

including the splicing of pre-mRNAs.
Ribozimas
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.
QuickTime™ and a
QuickTime™ and a
QuickTime™ and a
QuickTime™ and a
QuickTime™ and a
Aciclovir- a guanine derivative, DNA polymerase inhibitor and antiviral
QuickTime™ and a
Nucleótidos modificados
Pseudouridine (Ψ) was discovered to

be the most abundant
QuickTime™ and a
posttranscriptionally modified
nucleotide in cellular RNAs in the
1950s
QuickTime™ and a
DNA metiltransferasas

QuickTime™ and a
1950s
QuickTime™ and a
X
uracilo
desaminación espontánea de citosina,
se repara (DNA repair enzymes)

QuickTime™ and a
1950s
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
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 the
absorbance- hypochromic shift.
• When the two strands separate, this quenching disappears and the absorbance
rises 30-40% hyperchromic shift.
Denaturation of DNA Causes Hyperchromism
Tm: temperatura a la cual
Relative absorbance (260 nm)

el 50% del DNA es
cadena doble
Depende de factores como: 1.3
Adapted from Voet et al (1999) Fundamental of Biochemistry p.739

Composición nucleotídica
Entorno (fuerza iónica, pH,…)
Largo (complejidad) de las 1.2
cadenas a hibridar
Tm
1.1
1.0 50 70 90
Temperature (C)
G+C Content Is Proportional to Tm
Relative absorbance (260 nm)
Pneumococcus
(38%) G+C E. coli (52%)
1.4
S. Marcescens
Adapted from Garrett & Grisham (1999) Biochemistry (2e) p.372

(58%)
1.2
M. Phlei
(66%)
1.0
70 80 90 100
Temperature (C)
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
100
0.01 M phosphate
0.001 M EDTA 5’ 3’
80
G + C (%)
60
40
0.15 M NaCl
20 0.015 M Na citrate
0
5’ 3’
60 70 80 90 100 110
Tm (C)
Adapted from Garrett & Grisham (1999) Biochemistry (2e) p.372
Síntesis química de oligonucleótidos
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. ß-cianoethylphosphoramidite
4.Oxidación
Finalmente, el grupo fosfito
internucleotídico se oxida a
un grupo fosfato estable.
TAREA:
¿Qué es una topoisomerasa?
¿Tipos de topoisomerasa y cuál son sus funciones?

Nucleotidos 2

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Clase anterior:

* B-DNA is found at low salt concentrations. It is believed to be the native conformation

In order for base-pairing

In order for base-pairing

A triple-stranded DNA is a structure of DNA in which three oligonucleotides wind

DNA replication is a semi-conservative process.

Main types of RNAs

mRNAs messenger RNAs, code for proteins.

rRNAs ribosomal RNAs, components of the ribosome and catalyze

snRNAs small nuclear RNAs, function in a variety of nuclear processes,

Pseudouridine (Ψ) was discovered to

Pseudouridine (Ψ) was discovered to

Pseudouridine (Ψ) was discovered to

desaminación espontánea de 5-metil

• Lack an -OH group at

• Bases in DNA absorb light at 260 nm.

Relative absorbance (260 nm)

Depende de factores como: 1.3

Adapted from Voet et al (1999) Fundamental of Biochemistry p.739

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

Tamaño genoma (pb)

¿Qué es una topoisomerasa?

¿Tipos de topoisomerasa y cuál son sus funciones?

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